Sanjay Swarup

Glycine Decarboxylase Activity Drives Non-Small Cell Lung Cancer Tumor-Initiating Cells and Tumorigenesis

Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.

Enhancement of Plant-Microbe Interactions Using a Rhizosphere Metabolomics-Driven Approach and Its Application in the Removal of Polychlorinated Biphenyls

Persistent organic pollutants, such as polychlorinated biphenyls (PCBs), are a global problem. We demonstrate enhanced depletion of PCBs using root-associated microbes, which can use plant secondary metabolites, such as phenylpropanoids. Using a “rhizosphere metabolomics” approach, we show that phenylpropanoids constitute 84% of the secondary metabolites exuded from Arabidopsis roots. Phenylpropanoid-utilizing microbes are more competitive and are able to grow at least 100-fold better than their auxotrophic mutants on roots of plants that are able to synthesize or overproduce phenylpropanoids, such as flavonoids. Better colonization of the phenylpropanoid-utilizing strain in a gnotobiotic system on the roots of flavonoid-producing plants leads to almost 90% removal of PCBs in a 28-d period. Our work complements previous approaches to engineer soil microbial populations based on opines produced by transgenic plants and used by microbes carrying opine metabolism genes. The current approach based on plant natural products can be applied to contaminated soils with pre-existing vegetation. This strategy is also likely to be applicable to improving the competitive abilities of biocontrol and biofertilization strains.

The Cyclic Nucleotide Monophosphate Domain of Xanthomonas campestris Global Regulator Clp Defines a New Class of Cyclic Di-GMP Effectors

The widely conserved second messenger cyclic diguanosine monophosphate (c-di-GMP) plays a key role in quorum-sensing (QS)-dependent production of virulence factors in Xanthomonas campestris pv. campestris. The detection of QS diffusible signal factor (DSF) by the sensor RpfC leads to the activation of response regulator RpfG, which activates virulence gene expression by degrading c-di-GMP. Here, we show that a global regulator in the X. campestris pv. campestris QS regulatory pathway, Clp, is a c-di-GMP effector. c-di-GMP specifically binds to Clp with high affinity and induces allosteric conformational changes that abolish the interaction between Clp and its target gene promoter. Clp is similar to the cyclic AMP (cAMP) binding proteins Crp and Vfr and contains a conserved cyclic nucleotide monophosphate (cNMP) binding domain. Using site-directed mutagenesis, we found that the cNMP binding domain of Clp contains a glutamic acid residue (E99) that is essential for c-di-GMP binding. Substituting the residue with serine (E99S) resulted in decreased sensitivity to changes in the intracellular c-di-GMP level and attenuated bacterial virulence. These data establish the direct role of Clp in the response to fluctuating c-di-GMP levels and depict a novel mechanism by which QS links the second messenger with the X. campestris pv. campestris virulence regulon.

Berberine targets assembly of Escherichia coli cell division protein FtsZ.

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non-pathogenic Pseudomonas species.

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.

MorA Defines a New Class of Regulators Affecting Flagellar Development and Biofilm Formation in Diverse Pseudomonas Species

Assembly of bacterial flagella is developmentally important during both planktonic cell growth and biofilm formation. Flagellar biogenesis is complex, requiring coordinated expression of over 40 genes, and normally commences during the log-to-stationary transition phase. We describe here a novel membrane-localized regulator, MorA, that controls the timing of flagellar development and affects motility, chemotaxis, and biofilm formation in Pseudomonas putida. MorA is conserved among diverse Pseudomonas species, and homologues are present in all Pseudomonas genomes sequenced thus far. In P. putida, the absence of MorA derepresses flagellar development, which leads to constitutive formation of flagella in the mutant cells in all growth phases. In Pseudomonas aeruginosa, the absence of MorA led to a reduction in biofilm formation. However, unlike the motility of P. putida, the motility of the P. aeruginosa mutants was unaffected. Our data illustrate a novel developmentally regulated sensory and signaling pathway for several properties required for virulence and ecological fitness of Pseudomonas species.

Metabolomics and its role in understanding cellular responses in plants

A natural shift is taking place in the approaches being adopted by plant scientists in response to the accessibility of systems-based technology platforms. Metabolomics is one such field, which involves a comprehensive non-biased analysis of metabolites in a given cell at a specific time. This review briefly introduces the emerging field and a range of analytical techniques that are most useful in metabolomics when combined with computational approaches in data analyses. Using cases from Arabidopsis and other selected plant systems, this review highlights how information can be integrated from metabolomics and other functional genomics platforms to obtain a global picture of plant cellular responses. We discuss how metabolomics is enabling large-scale and parallel interrogation of cell states under different stages of development and defined environmental conditions to uncover novel interactions among various pathways. Finally, we discuss selected applications of metabolomics.

Elucidation of the Flavonoid Catabolism Pathway in Pseudomonas putida PML2 by Comparative Metabolic Profiling

ABSTRACT Flavonoids are 15-carbon plant secondary metabolites exuded in the rhizosphere that hosts several flavonoid-degrading bacteria. We studied flavonoid catabolism in a plant growth-promoting rhizobacterial strain of Pseudomonas by using a combination of biochemical and genetic approaches. Transposants carrying mini-Tn5gfp insertions were screened for flavonoid auxotrophy, and these mutant strains were found to be unable to grow in the flavonols naringenin and quercetin, while their growth in glycerol was comparable to that of the parental strain. In order to understand flavonoid catabolism, culture supernatants, whole-cell fractions, cell lysate, and cell debris of the wild-type and mutant strains were analyzed. Intermediates that accumulated intracellularly and those secreted in the medium were identified by a combination of reversed-phase high-pressure liquid chromatography and electrospray ionization-mass spectrometry. Structures of four key intermediates were confirmed by one-dimensional nuclear magnetic resonance spectroscopy. Comparative metabolic profiling of the compounds in the wild-type and mutant strains allowed us to understand the degradation events and to identify six metabolic intermediates. The first step in the pathway involves 3,3′-didehydroxylation, followed by hydrolysis and cleavage of the C-ring, leading via subsequent oxidations to the formation of protocatechuate. This is the first report on quercetin dehydroxylation in aerobic conditions leading to naringenin accumulation.

Quorum sensing modulation of a putative glycosyltransferase gene cluster essential for Xanthomonas campestris biofilm formation

Findings from previous studies suggest that the quorum sensing signal DSF (diffusible signal factor) negatively regulates biofilm formation in Xanthomonas campestris pv. campestris (Xcc) by affecting the expression of manA encoding biofilm dispersion and an unknown factor(s). In this study, by analysing the double deletion mutant ΔrpfFΔmanA, in which DSF biosynthesis gene rpfF and biofilm dispersal gene manA were deleted, we found that DSF modulated biofilm development by suppression of a mechanism essential for biofilm formation. Transposon mutagenesis of ΔrpfFΔmanA and subsequent analyses led to the identification of a novel gene locus xagABC encoding a putative glycosyl transferase system. Genetic analysis revealed that the transcriptional expression of xagABC was negatively regulated by DSF through the RpfC/RpfG two-component regulatory system. Deletion of the xag genes resulted in decreased extracellular polysaccharide production, abolished Xcc biofilm formation and attenuated the bacterial resistance to oxidative stress. Furthermore, we provide evidence that xagABC and manA were differentially expressed in Xcc and the biofilm formed by overexpression of xagABC in wild-type Xcc could be dispersed by ManA. These results provide new insight into the molecular mechanisms by which Xcc switches between planktonic growth and biofilm lifestyle.

Synthesis and in vitro antiproliferative activity of novel 1-benzhydrylpiperazine derivatives against human cancer cell lines

In order to explore the antiproliferative effect associated with the piperazine framework, several 1-benzhydrylpiperazine derivatives 8(a-d), 9(a-d) and 10(a-h) were synthesized. Variation in the functional group at N-terminal of the piperazine led to three sets of compounds, bearing the sulfonyl, amide and thiourea, respectively. Their chemical structures were confirmed by (1)H NMR, LCMS, IR and elemental analysis. The antiproliferative effect of the compounds were evaluated in vitro using the MTT colorimetric method against one normal cell line (NF-103 skin fibroblast cells) and four human cancer cell lines (MCF-7 breast carcinoma cell line, HepG-2 hepatocellular carcinoma cell line, HeLa cervix carcinoma cell line and HT-29 colon carcinoma cell line) for the time period of 24 h. Among the series, four compounds exhibited interesting growth inhibitory effects against all four cell lines.