Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis,and Concurrently Affect the Pathogen's Growth

Background

The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung.

Methodology/Principal Findings

During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.

Conclusions/Significance

While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide''s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.     

Novel diphenyl ethers: design, docking studies, synthesis and inhibition of enoyl ACP reductase of Plasmodium falciparum and Escherichia coli

We designed some novel diphenyl ethers and determined their binding energies for Enoyl-Acyl Carrier Protein Reductase (ENR) of Plasmodium falciparum using Autodock. Out of these, we synthesized the promising compounds and tested them for their inhibitory activity against ENRs of P. falciparum as well as Escherichia coli. Some of these compounds show nanomolar inhibition of PfENR and low micromolar inhibition of EcENR. They also exhibit low micromolar potency against in vitro cultures of P. falciparum and E. coli. The study of structure-activity relationship of these compounds paves the way for further improvements in the design of novel diphenyl ethers with improved activity against purified enzyme and the pathogens.     

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.     

Modification of chromium (VI) phytotoxicity by exogenous gibberellic acid application in <Emphasis Type="Italic">Pisum sativum</Emphasis> (L.) seedlings

Effects of exogenous gibberellic acid (GA; 10 and 100 μM) application on growth, protein and nitrogen contents, ammonium (NH₄ ⁺) content, enzymes of nitrogen assimilation and antioxidant system in pea seedlings were investigated under chromium (VI) phytotoxicity (Cr VI; 50, 100 and 250 μM). Exposure of pea seedlings to Cr and 100 μM GA resulted in decreased seed germination, fresh and dry weight and length of root and shoot, and protein and nitrogen contents compared to control. Compared to control, Cr and 100 μM GA led to the significant alteration in nitrogen assimilation in pea. These treatments decreased root and shoot nitrate reductase (NR), glutamine synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT) activities (except 50 μM Cr alone for GOGAT) while glutamate dehydrogenase (GDH) activity and NH₄ ⁺ content increased. Compared to control, the root and shoot activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased (except APX activity at 250 μM Cr + 100 μM GA) while catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) activities were decreased (except GR at 100 μM GA alone) following exposure of Cr and 100 μM GA. Total ascorbate and total glutathione in root and shoot decreased by the treatments of Cr and 100 μM GA while their levels were increased by the application of 10 μM GA compared to Cr treatments alone. It has been reported that application of 10 μM GA together with Cr alleviated inhibited levels of growth, nitrogen assimilation and antioxidant system compared to Cr treatments alone. This study showed that application of 10 μM GA counteracts some of the adverse effects of Cr phytotoxicity with the increased levels of antioxidants and sustained activities of enzymes of nitrogen assimilation; however, 100 μM GA showed apparently reverse effect under Cr phytotoxicity.     

A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don

An efficient and cost effective micropropagation protocol using liquid medium was developed for Catharanthus roseus, a commercially important medicinal plant. Comparative analysis of shoot growth and proliferation in liquid Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins [6-Benzyladenine (BA), Kinetin (KN) and Thidiazuron (TDZ)] was conducted. Better response in terms of shoot proliferation, shoot diameter, number of leaves/shoot, number of branches/shoot, fresh weight and dry weight was observed in a liquid medium vis-à-vis solid medium. A sample of 20 ml of liquid medium supplemented with 5 ??M of BA was optimized for propagation of C. roseus by a liquid culture system. Among various concentrations of auxins tried, 1-Naphthaleneacetic acid (NAA) 5 ??M was found to be the best for root induction. Quantification of pharmaceutically important constituents (vincristine and vinblastine) and total alkaloid content of microshoots grown in solid and liquid medium as well as in vitro raised plants and mother plant was also conducted, hitherto unreported in this high-value medicinal plant. This work further lays the foundations for the shifting of plant production from small to commercial scale.     

Structural insights into the aggregation behavior of Murraya koenigii miraculin‐like protein below pH 7.5

Murraya koenigii miraculin‐like protein (MKMLP) gradually precipitates below pH 7.5. Here, we explore the basis for this aggregation by identifying the aggregation‐prone regions via comparative analysis of crystal structures acquired at several pH values. The prediction of aggregation‐prone regions showed the presence of four short peptides either in beta sheets or loops on surface of the protein. These peptides were distributed in two patches far apart on the surface. Comparison of crystal structures of MKMLP, determined at 2.2 Å resolution in pH 7.0 and 4.6 in the present study and determined at 2.9 Å in pH 8.0 in an earlier reported study, reveal subtle conformational differences resulting in gradual exposure of aggregation‐prone regions. As the pH is lowered, there are alterations in ionic interactions within the protein interactions of the chain with water molecules and exposure of hydrophobic residues. The analysis of symmetry‐related molecular interfaces involving one patch revealed shortening of nonpolar intermolecular contacts as the pH decreased. In particular, a decrease in the intermolecular distance between Trp103 of the aggregation‐prone peptide WFITTG (103–108) unique to MLPs was observed. These results demonstrated that aggregation occurs due to the cumulative effect of the changes in interactions in two aggregation‐prone defined regions. Proteins 2014; 82:830–840. © 2013 Wiley Periodicals, Inc.