Competition between H4PteGlu and H2PtePAS Confers para-Aminosalicylic Acid Resistance in Mycobacterium tuberculosis.

Tuberculosis remains a serious challenge to human health worldwide. para-Aminosalicylic acid (PAS) is an important anti-tuberculosis drug, which requires sequential activation by Mycobacterium tuberculosis (M. tuberculosis) dihydropteroate synthase and dihydrofolate synthase (DHFS, FolC). Previous studies showed that loss of function mutations of a thymidylate synthase coding gene thyA caused PAS resistance in M. tuberculosis, but the mechanism is unclear. Here we showed that deleting thyA in M. tuberculosis resulted in increased content of tetrahydrofolate (H4PteGlu) in bacterial cells as they rely on the other thymidylate synthase ThyX to synthesize thymidylate, which produces H4PteGlu during the process. Subsequently, data of in vitro enzymatic activity experiments showed that H4PteGlu hinders PAS activation by competing with hydroxy dihydropteroate (H2PtePAS) for FolC catalysis. Meanwhile, over-expressing folC in ΔthyA strain and a PAS resistant clinical isolate with known thyA mutation partially restored PAS sensitivity, which relieved the competition between H4PteGlu and H2PtePAS. Thus, loss of function mutations in thyA led to increased H4PteGlu content in bacterial cells, which competed with H2PtePAS for catalysis by FolC and hence hindered the activation of PAS, leading to decreased production of hydroxyl dihydrofolate (H2PtePAS-Glu) and finally caused PAS resistance. On the other hand, functional deficiency of thyA in M. tuberculosis pushes the bacterium switch to an unidentified dihydrofolate reductase for H4PteGlu biosynthesis, which might also contribute to the PAS resistance phenotype. Our study revealed how thyA mutations confer PAS resistance in M. tuberculosis and provided new insights into studies on the folate metabolism of the bacterium.

CycA-Dependent Glycine Assimilation Is Connected to Novobiocin Susceptibility in Escherichia coli.

Escherichia coli serine hydroxymethyltransferase (GlyA) converts serine to glycine, and glyA mutants are auxotrophic for glycine. CycA is a transporter that mediates glycine uptake. Deleting glyA in E. coli strain W3110 led to activation of CysB, which was related to novobiocin (NOV) susceptibility. Moreover, deleting glyA resulted in increased sensitivity to NOV, and this could be reversed by high concentrations of glycine. Reverse mutants of ΔglyA were selected and one of them had a mutation in yrdC, the gene encoding threonylcarbamoyl-AMP synthase. Subsequent proteome analysis showed that deleting glyA led to increased expression of TcyP and TdcB, making this bacterium dependent on CycA for glycine assimilation. Furthermore, deleting cycA in a ΔglyA background caused a severe growth defect on Luria-Bertani medium, which could be complemented by high concentrations of exogenous glycine. Mutation of yrdC led to decreased expression of TdcB but increased expression of ThrA/B/C and LtaE, which favored the conversion of threonine to glycine and thus avoided the dependence on CycA. Correspondingly, deleting of tcyP, tdcB, or gshA could reverse the NOV-sensitive phenotype of ΔglyA mutants. Overexpression of cycA resulted in increased sensitivity to NOV, whereas deleting this gene caused NOV resistance. Moreover, overexpression of cycA led to increased accumulation of NOV upon drug treatment. Therefore, inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which enhanced the accumulation of NOV and then made the bacterium more sensitive to this drug. These findings broaden our understanding of glycine metabolism and mechanisms of NOV susceptibility. IMPORTANCE Novobiocin (NOV) has been used in clinical practice as an ATPase inhibitor for decades. However, because it has been withdrawn from the market, pharmaceutical companies are searching for other ATPase inhibitors. Thus, probing the mechanisms of susceptibility to NOV will be beneficial to those efforts. In this study, we showed that inactivation of glyA in E. coli led to CycA-dependent glycine assimilation, which accompanied the accumulation of NOV and thereby increased the sensitivity to this drug. To date, this is the first report demonstrating the linkage between glycine assimilation and NOV susceptibility, and it is also the first report showing that YrdC is able to modulate the metabolic flux of threonine.

Decreased Methylenetetrahydrofolate Reductase Activity Leads to Increased Sensitivity to para-Aminosalicylic Acid in Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most fatal diseases in the world. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the production of 5-methyltetrahydrofolate (5-CH3-THF), which is required for the de novo biosynthesis of methionine in bacteria. Here, we identified Rv2172c as an MTHFR in M. tuberculosis through in vitro and in vivo analyses and determined that the protein is essential for the in vitro growth of the bacterium. Subsequently, we constructed rv2172c R159N and L214A mutants in M. tuberculosis and found that these mutants were more sensitive to the antifolates para-aminosalicylic acid (PAS) and sulfamethoxazole (SMX). Combining biochemical and genetic methods, we found that rv2172c R159N or L214A mutation impaired methionine production, leading to increased susceptibility of M. tuberculosis to PAS, which was largely restored by adding exogenous methionine. Moreover, overexpression of rv2172c in M. tuberculosis could increase methionine production and lead to PAS resistance. This research is the first to identify an MTHFR in M. tuberculosis and reveals that the activity of this enzyme is associated with susceptibility to antifolates. These findings have particular value for antitubercular drug design for the treatment of drug-resistant TB.

MgrB Inactivation Confers Trimethoprim Resistance in Escherichia coli.

After several decades of use, trimethoprim (TMP) remains one of the key access antimicrobial drugs listed by the World Health Organization. To circumvent the problem of trimethoprim resistance worldwide, a better understanding of drug-resistance mechanisms is required. In this study, we screened the single-gene knockout library of Escherichia coli, and identified mgrB and other several genes involved in trimethoprim resistance. Subsequent comparative transcriptional analysis between ΔmgrB and the wild-type strain showed that expression levels of phoP, phoQ, and folA were significantly upregulated in ΔmgrB. Further deleting phoP or phoQ could partially restore trimethoprim sensitivity to ΔmgrB, and co-overexpression of phoP/Q caused TMP resistance, suggesting the involvement of PhoP/Q in trimethoprim resistance. Correspondingly, MgrB and PhoP were shown to be able to modulated folA expression in vivo. After that, efforts were made to test if PhoP could directly modulate the expression of folA. Though phosphorylated PhoP could bind to the promotor region of folA in vitro, the former only provided a weak protection on the latter as shown by the DNA footprinting assay. In addition, deleting the deduced PhoP box in ΔmgrB could only slightly reverse the TMP resistance phenotype, suggesting that it is less likely for PhoP to directly modulate the transcription of folA. Taken together, our data suggested that, in E. coli, MgrB affects susceptibility to trimethoprim by modulating the expression of folA with the involvement of PhoP/Q. This work broadens our understanding of the regulation of folate metabolism and the mechanisms of TMP resistance in bacteria.

Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA.

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.

Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis.

Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA.

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity.

Mycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, Mycobacterium smegmatis, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis.

Structural analysis of the regulatory mechanism of MarR protein Rv2887 in M. tuberculosis.

MarR family proteins are transcriptional regulators that control expression of bacterial proteins involved in metabolism, virulence, stress responses and multi-drug resistance, mainly via ligand-mediated attenuation of DNA binding. Greater understanding of their underlying regulatory mechanism may open up new avenues for the effective treatment of bacterial infections. To gain molecular insight into the mechanism of Rv2887, a MarR family protein in M. tuberculosis, we first showed that it binds salicylate (SA) and para-aminosalicylic acid (PAS), its structural analogue and an antitubercular drug, in a 1:1 stoichiometry with high affinity. Subsequent determination and analysis of Rv2887 crystal structures in apo form, and in complex with SA, PAS and DNA showed that SA and PAS bind to Rv2887 at similar sites, and that Rv2887 interacts with DNA mainly by insertion of helix α4 into the major groove. Ligand binding triggers rotation of the wHTH domain of Rv2887 toward the dimerization domain, causing changes in protein conformation such that it can no longer bind to a 27 bp recognition sequence in the upstream region of gene Rv0560c. The structures provided here lay a foundation for the design of small molecules that target Rv2887, a potential new approach for the development of anti-mycobacterials.

Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide.

Tuberculosis is still on the top of infectious diseases list on both mobility and mortality, especially due to drug-resistance of Mycobacterium tuberculosis (M.tb). Ethionamide (ETH) is one of effective second line anti-TB drugs, a synthetic compound similar to isoniazid (INH) structurally, with existing severe problem of ETH resistance. ETH is a prodrug, which is activated by Etha inside M.tb, and etha is transcriptionally repressed by Ethr. We found that c-di-GMP could bind Ethr, enhanced the binding of Ethr to the promoter of etha, and then repressed the transcription of etha, thus caused resistance of M.tb to ETH. Through docking analysis and in vitro validation, we identified that c-di-GMP binds 3 amino acids of Ethr, i.e., Q125, R181 and E190, while the first 2 were the major binding sites. Homology analysis showed that Ethr was highly conservative among mycobacteria. Further docking analysis showed that c-di-GMP preferentially bound proteins of TetR family at the junction hole of symmetric dimer or tetramer proteins. Our results suggest a possible drug-resistance mechanism of ETH through the regulation of Ethr by c-di-GMP.

Deletion of sigB Causes Increased Sensitivity to para-Aminosalicylic Acid and Sulfamethoxazole in Mycobacterium tuberculosis.

Although the de novo folate biosynthesis pathway has been well studied in bacteria, little is known about its regulation. In the present study, the sigB gene in Mycobacterium tuberculosis was deleted. Subsequent drug susceptibility tests revealed that the M. tuberculosis ΔsigB strain was more sensitive to para-aminosalicylic acid (PAS) and sulfamethoxazole. Comparative transcriptional analysis was performed, and downregulation of pabB was observed in the ΔsigB strain, which was further verified by a quantitative reverse transcription-PCR and Western blot assay. Then, the production levels of para-aminobenzoic acid (pABA) were compared between the sigB deletion mutant and wild-type strain, and the results showed that sigB deletion resulted in decreased production of pABA. In addition, SigB was able to recognize the promoter of pabB in vitro Furthermore, we found that deleting pabC also caused increased susceptibility to PAS. Taken together, our data revealed that, in M. tuberculosis, sigB affects susceptibility to antifolates through multiple ways, primarily by regulating the expression of pabB To our knowledge, this is the first report showing that SigB modulates pABA biosynthesis and thus affecting susceptibility to antifolates, which broadens our understanding of the regulation of bacterial folate metabolism and mechanisms of susceptibility to antifolates.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.

Deletion of nudB Causes Increased Susceptibility to Antifolates in Escherichia coli and Salmonella enterica.

Co-trimoxazole, a fixed-dose combination of sulfamethoxazole (SMX) and trimethoprim (TMP), has been used for the treatment of bacterial infections since the 1960s. Since it has long been assumed that the synergistic effects between SMX and TMP are the consequence of targeting 2 different enzymes of bacterial folate biosynthesis, 2 genes (pabB and nudB) involved in the folate biosynthesis of Escherichia coli were deleted, and their effects on the susceptibility to antifolates were tested. The results showed that the deletion of nudB resulted in a lag of growth in minimal medium and increased susceptibility to both SMX and TMP. Moreover, deletion of nudB also greatly enhanced the bactericidal effect of TMP. To elucidate the mechanism of how the deletion of nudB affects the bacterial growth and susceptibility to antifolates, 7,8-dihydroneopterin and 7,8-dihydropteroate were supplemented into the growth medium. Although those metabolites could restore bacterial growth, they had no effect on susceptibilities to the antifolates. Reverse mutants of the nudB deletion strain were isolated to further study the mechanism of how the deletion of nudB affects susceptibility to antifolates. Targeted sequencing and subsequent genetic studies revealed that the disruption of the tetrahydromonapterin biosynthesis pathway could reverse the phenotype caused by the nudB deletion. Meanwhile, overexpression of folM could also lead to increased susceptibility to both SMX and TMP. These data suggested that the deletion of nudB resulted in the excess production of tetrahydromonapterin, which then caused the increased susceptibility to antifolates. In addition, we found that the deletion of nudB also resulted in increased susceptibility to both SMX and TMP in Salmonella enterica Since dihydroneopterin triphosphate hydrolase is an important component of bacterial folate biosynthesis and the tetrahydromonapterin biosynthesis pathway also exists in a variety of bacteria, it will be interesting to design new compounds targeting dihydroneopterin triphosphate hydrolase, which may inhibit bacterial growth and simultaneously potentiate the antimicrobial activities of antifolates targeting other components of folate biosynthesis.

Anti-folates potentiate bactericidal effects of other antimicrobial agents.

Synergies between sulfonamides and other antimicrobial agents have long been reported, but the reason still remains unclear. Previously, Vilchèze et al. found that, sulfamethoxazole (SMX) could potentiate the bacterialcidal activity of isoniazid (INH) and rifampin (RIF) in Mycobacterium tuberculosis. To test if this was also the case in other bacteria, the ability to potentiate bactericidal effect of RIF by SMX was evaluated in Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Mycobacterium smegmatis. And the ability to potentiate bactericidal effect of streptomycin (SM) by SMX was also evaluated in E. coli and M. Smegmatis. Susceptibility tests and drug exposure experiments were performed for RIF and SM in the presence of sub-ICs of SMX. In drug exposure experiments, 10 mg l-1 of 7,8-dihydropteroic acid (DHP) was used to reverse the effect of SMX. In the presence of sub-ICs of SMX, MIC of RIF for E. coli and M. smegmatis decreased 2 and 16 fold, respectively. In the drug exposure experiments, addition of sub-ICs of SMX suppressed the growth of RIF and SM resistant population in a pool of susceptible bacteria, and the effects of SMX could be reversed by DHP. Besides, we also found that, sub-ICs of para-aminosalicylic acid (PAS) could bactericidal effects of INH, RIF and SM in M. tuberculosis. Taken together, our data suggest that, sub-ICs of anti-folates can potentiate bactericidal effects of other antimicrobial agents in various bacteria.

Synthesis of adenosine-imprinted microspheres for the recognition of ADP-ribosylated proteins.

Core-shell structural adenosine-imprinted microspheres were prepared via a two-step procedure. Polystyrene core particles (CP) were firstly prepared via a reversible addition-fragmentation chain transfer (RAFT) polymerization leaving the iniferter on the surface of the cores, then a molecularly imprinted polymer (MIP) shell was synthesized on the surface of the cores by using acrylamide (AAm) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The formation and growth of the MIP layer were seen dependent on the initiator (AIBN), AAm and the polymerization time used within the polymerization. SEM/TEM images showed that the dimensions of the cores and shells were 2µM and 44nm, respectively. The MIP microspheres exhibited a fast rebinding rate within 2h and a maximum adsorption capacity of 177µg per gram for adenosine. The adsorption fitted a Langmuir-Freundlich (LF) isotherm model with a KLF value of 41mL/µg and a qm value of 177µg/g for the MIP microspheres. The values were larger than those for a non-molecularly imprinted polymer (NIP) particles (5mL/µg and 88µg/g) indicating a better adsorption ability towards adenosine. The MIP microspheres showed a good selectivity for adenosine with a higher adsorption (683nmol/g) for adenosine than that (91nmol/g, 24nmol/g and 54nmol/g) for guanosine, cytidine and uridine respectively. Further experiment proved that the adenosine-imprinted polymer microspheres also had a good selectivity for ADP-ribosylated proteins that the MIP could extract the ADP-ribosylated proteins from the cell extract samples.

YcgC represents a new protein deacetylase family in prokaryotes.

Reversible lysine acetylation is one of the most important protein posttranslational modifications that plays essential roles in both prokaryotes and eukaryotes. However, only a few lysine deacetylases (KDACs) have been identified in prokaryotes, perhaps in part due to their limited sequence homology. Herein, we developed a 'clip-chip' strategy to enable unbiased, activity-based discovery of novel KDACs in the Escherichia coli proteome. In-depth biochemical characterization confirmed that YcgC is a serine hydrolase involving Ser200 as the catalytic nucleophile for lysine deacetylation and does not use NAD(+) or Zn(2+) like other established KDACs. Further, in vivo characterization demonstrated that YcgC regulates transcription by catalyzing deacetylation of Lys52 and Lys62 of a transcriptional repressor RutR. Importantly, YcgC targets a distinct set of substrates from the only known E. coli KDAC CobB. Analysis of YcgC's bacterial homologs confirmed that they also exhibit KDAC activity. YcgC thus represents a novel family of prokaryotic KDACs.

Self-Assembly of Ferritin Nanoparticles into an Enzyme Nanocomposite with Tunable Size for Ultrasensitive Immunoassay.

The self-assembly of nanoparticles into larger superstructures is a powerful strategy to develop novel functional nanomaterials, as these superstructures display collective properties that are different to those displayed by individual nanoparticles or bulk samples. However, there are increasing bottlenecks in terms of size control and multifunctionalization of nanoparticle assemblies. In this study, we developed a self-assembly strategy for construction of multifunctional nanoparticle assemblies of tunable size, through rational regulation of the number of self-assembling interaction sites on each nanoparticle. As proof-of-principle, a size-controlled enzyme nanocomposite (ENC) was constructed by self-assembly of streptavidin-labeled horseradish peroxidase (SA-HRP) and autobiotinylated ferritin nanoparticles (bFNP). Our ENC integrates a large number of enzyme molecules, together with a streptavidin-coated surface, allowing for a drastic increase in enzymatic signal when the SA is bound to a biotinylated target molecule. As result, a 10 000-fold increase in sensitivity over conventional enzyme-linked immunosorbent assays (ELISA) methods was achieved in a cardiac troponin immunoassay. Our method presented here should provide a feasible approach for constructing elaborate multifunctional superstructures of tunable size useful for a broad range of biomedical applications.

The S28H mutation on mNeptune generates a brighter near-infrared monomeric fluorescent protein with improved quantum yield and pH-stability.

For living deep-tissue imaging, the optical window favorable for light penetration is in near-infrared wavelengths, which requires fluorescent proteins with emission spectra in the near-infrared region. Here, we report that a single mutant Ser28His of mNeptune with a near-infrared (≥650 nm) emission maxima of 652 nm is found to improve the brightness, photostability, and pH stability when compared with its parental protein mNeptune, while it remains as a monomer, demonstrating that there is still plenty of room to improve the performance of the existing near infrared fluorescence proteins by directed evolution.

Binding pocket alterations in dihydrofolate synthase confer resistance to para-aminosalicylic acid in clinical isolates of Mycobacterium tuberculosis.

The mechanistic basis for the resistance of Mycobacterium tuberculosis to para-aminosalicylic acid (PAS), an important agent in the treatment of multidrug-resistant tuberculosis, has yet to be fully defined. As a substrate analog of the folate precursor para-aminobenzoic acid, PAS is ultimately bioactivated to hydroxy dihydrofolate, which inhibits dihydrofolate reductase and disrupts the operation of folate-dependent metabolic pathways. As a result, the mutation of dihydrofolate synthase, an enzyme needed for the bioactivation of PAS, causes PAS resistance in M. tuberculosis strain H37Rv. Here, we demonstrate that various missense mutations within the coding sequence of the dihydropteroate (H2Pte) binding pocket of dihydrofolate synthase (FolC) confer PAS resistance in laboratory isolates of M. tuberculosis and Mycobacterium bovis. From a panel of 85 multidrug-resistant M. tuberculosis clinical isolates, 5 were found to harbor mutations in the folC gene within the H2Pte binding pocket, resulting in PAS resistance. While these alterations in the H2Pte binding pocket resulted in reduced dihydrofolate synthase activity, they also abolished the bioactivation of hydroxy dihydropteroate to hydroxy dihydrofolate. Consistent with this model for abolished bioactivation, the introduction of a wild-type copy of folC fully restored PAS susceptibility in folC mutant strains. Confirmation of this novel PAS resistance mechanism will be beneficial for the development of molecular method-based diagnostics for M. tuberculosis clinical isolates and for further defining the mode of action of this important tuberculosis drug.

Acetylation reduces the ability of CheY to undergo autophosphorylation.

CheY, the response regulator of the chemotaxis system in Escherichia coli, can be regulated by two covalent modifications - phosphorylation and acetylation. Both covalent modifications are involved in chemotaxis, but the mechanism and role of the acetylation are still obscure. While acetylation was shown to repress the binding of CheY to its target proteins, the effect of acetylation on the ability of CheY to undergo autophosphorylate with AcP is not fully investigated. To obtain more information on the function of this acetylation, we successfully expressed and purified CheY protein with a 6 × His-tag on the C-terminus. Subsequently, acetylated CheY (AcCheY) was obtained with AcCoA as the acetyl donor, and the acetylation level of AcCheY was confirmed by Western blotting and then mass spectrometry. Using tryptophan fluorescence intensity measurements as a monitor of phosphorylation, we showed that acetylation reduces the ability of CheY to undergo autophosphorylation.

Quantum dot-induced viral capsid assembling in dissociation buffer.

Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs) are still unknown. In this article, it was found that quantum dots (QDs) can induce simian virus 40 (SV40) capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1) can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD=2.19E-10 M), which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles.