Application of the codon-shuffling method : Synthesis and selection of de novo proteins as antibacterials

Library-based methods of non-rational and part-rational designed de novo peptides are worthy beacons in the search for bioactive peptides and proteins of medicinal importance. In this report, we have used a recently developed directed evolution method called "codon shuffling" for the synthesis and selection of bioactive proteins. The selection of such proteins was based on the creation of an inducible library of "codon-shuffled" genes that are constructed from the ligation-based assembly of judiciously designed hexamer DNA duplexes called dicodons. Upon induction with isopropyl 1-thio-beta-D-galactopyranoside, some library members were found to express dicodon-incorporated proteins. Because of this, the host cells, in our case Escherichia coli, were unable to grow any further. The bactereostatic/lytic nature of the dicodon proteins was monitored by growth curves as well as by zone clearance studies. Transmission electron microscopy of the affected cells illustrated the extent of cell damage. The proteins themselves were overexpressed as fusion partners and subsequently purified to homogeneity. One such purified protein was found to strongly bind heparin, an indication that the interaction of the de novo proteins may be with the nucleic acids of the host cell, much like many of the naturally occurring antibacterial peptides, e.g. Buforin. Therefore, our approach may help in generating a multitude of finely tuned antibacterial proteins that can potentially be regarded as lead compounds once the method is extended to pathogenic hosts, such as Mycobacteria, for example

EphH, a unique epoxide hydrolase encoded by Rv3338 is involved in the survival of Mycobacterium tuberculosis under in vitro stress and vacuolar pH-induced changes

IntroductionMycobacterium tuberculosis (Mtb), one of the deadliest human pathogen, has evolved with different strategies of survival inside the host, leading to a chronic state of infection. Phagosomally residing Mtb encounters a variety of stresses, including increasing acidic pH. To better understand the host-pathogen interaction, it is imperative to identify the role of various genes involved in the survivability of Mtb during acidic pH environment.MethodsBio-informatic and enzymatic analysis were used to identify Mtb gene, Rv3338, as epoxide hydrolase. Subsequently, CRISPRi knockdown strategy was used to decipher its role for Mtb survival during acidic stress, nutrient starvation and inside macrophages. Confocal microscopy was used to analyse its role in subverting phagosomal acidification within macrophage.ResultsThe present work describes the characterization of Rv3338 which was previously known to be associated with the aprABC locus induced while encountering acidic stress within the macrophage. Bio-informatic analysis demonstrated its similarity to epoxide hydrolase, which was confirmed by enzymatic assays, thus, renamed EphH. Subsequently, we have deciphered its indispensable role for Mtb in protection from acidic stress by using the CRISPRi knockdown strategy. Our data demonstrated the pH dependent role of EphH for the survival of Mtb during nutrient starvation and in conferring resistance against elevated endogenous ROS levels during stress environment.ConclusionTo the best of our knowledge, this is the first report of an EH of Mtb as a crucial protein for bacterial fitness inside the host, a phenomenon central to its pathogenesis

Drug discovery targeting drug-resistant bacteria [electronic resource] /

Includes index.Elsevie

A comparative study on the isolation and cerium (IV) ammonium nitrate mediated oxidative transformation of oleanolic acid and ursolic acid

598-603<span style="font-size:11.0pt;mso-bidi-font-size: 10.0pt;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-bidi-font-family:"times="" roman";letter-spacing:-.1pt;mso-ansi-language:="" en-gb;mso-fareast-language:en-us;mso-bidi-language:ar-sa"="" lang="EN-GB">Oleanolic acid and Ursolic acid, two important pentacyclic triterpenes have been extracted from two different sources and have been subjected to oxidation using cerium (IV) ammonium nitrate in methanol. Two new oxidation products, namely a lactone and an ester have been obtained which however show decreased antibacterial activity in comparison to the parent compounds.</span

Investigating the Photosensitivity of Koneramines for Cell Imaging and Therapeutic Applications

The photophysical properties of the anthracene appended koneramines (LAn) were analyzed and utilized as a chemosensor for the selective detection of Cd2+ and Zn2+. The complexation-induced inhibition of PET (photo-induced electron transfer) from the chelating nitrogen atoms to the excited state of the anthracene moiety resulted in a fluorescence "turn-on" signal upon binding with Cd2+ and Zn2+. The confocal microscopic imaging studies performed on the MCF-7 cells validated that the compound is potentially useful for detecting Cd2+ and Zn2+ inside the cells. The cadmium complex exhibited unique bactericidal activity against clinically relevant human pathogens. The excellent activity against multidrug-resistant S. aureus makes the complex useful as a new, easily synthesizable antibiotic. The cadmium complex LAnCdCl2 was not cytotoxic against vero cells with a selectivity index of 40, exhibited concentration dependent bactericidal killing, non-interactive with several other clinically approved standard drugs, exhibited prolonged post-antibiotic effect (PAE) against S. aureus ATCC 29213 and possesses antibiofilm activity

Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide ‘SVAP9I’ that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4–8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections