In Silico Identification of Novel Derivatives of Rifampicin Targeting Ribonuclease VapC2 of M. tuberculosis H37Rv: Rifampicin Derivatives Target VapC2 of Mtb H37Rv.

The emergence of multi-drug-resistant Mycobacterium tuberculosis (Mtb) strains has rendered many of the currently available anti-TB drugs ineffective. Hence, there is a pressing need to discover new potential drug targets/candidates. In this study, attempts have been made to identify novel inhibitors of the ribonuclease VapC2 of Mtb H37Rv using various computational techniques. Ribonuclease VapC2 Mtb H37Rv's protein structure was retrieved from the PDB databank, 22 currently used anti-TB drugs were retrieved from the PubChem database, and protein-ligand interactions were analyzed by docking studies. Out of the 22 drugs, rifampicin (RIF), being a first-line drug, showed the best binding energy (-8.8 Kcal/mol) with Mtb H37Rv VapC2; hence, it was selected as a parent molecule for the design of its derivatives. Based on shape score and radial plot criteria, out of 500 derivatives designed through SPARK (Cresset®, Royston, UK) program, the 10 best RIF derivatives were selected for further studies. All the selected derivatives followed the ADME criteria concerning drug-likeness. The docking of ribonuclease VapC2 with RIF derivatives revealed the best binding energy of -8.1 Kcal/mol with derivative 1 (i.e., RIF-155841). A quantitative structure-activity relationship study revealed that derivative 1's activity assists in the inhibition of ribonuclease VapC2. The stability of the VapC2-RIF155841 complex was evaluated using molecular dynamics simulations for 50 ns and the complex was found to be stable after 10 nsec. Further, a chemical synthesis scheme was designed for the newly identified RIF derivative (RIF-155841), which verified that its chemical synthesis is possible for future in vitro/in vivo experimental validation. Overall, this study evaluated the potential of the newly designed RIF derivatives with respect to the Mtb VapC2 protein, which is predicted to be involved in some indispensable processes of the related pathogen. Future experimental studies regarding RIF-155841, including the exploration of the remaining RIF derivatives, are warranted to verify our current findings.

Engineering a Carotenoid-Overproducing Strain of Azospirillum brasilense for Heterologous Production of Geraniol and Amorphadiene.

Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

RelQ Mediates the Expression of ß-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus.

An induced stringent response, which is established by an increased level of (p)ppGpp, is required for the expression of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). However, it is not clear whether RSH (enzyme mediating stringent response to amino acid starvation) or small alarmone synthetases (SASs) are involved in the maintenance of (p)ppGpp level in response to ß-lactams. Since the S. aureus genome encodes two active SASs (RelP and RelQ), their contribution to the expression of ß-lactam resistance in MRSA was investigated. It was determined that relQ deletion renders community-associated MRSA (CA-MRSA) sensitive to ß-lactams by negatively affecting the expression of mecA, and induction of (p)ppGpp synthesis by mupirocin bypasses the requirement of relQ for the expression of high-level ß-lactam resistance. Surprisingly, relP deletion increased the level of ß-lactam resistance. Such contradictory observations could be attributed to the fact that relQ promoter is ~5-fold stronger than the relP and is induced by oxacillin as well as deletion of either of the SASs, while relP promoter responds only to oxacillin. The stronger promoter activity of relQ, coupled with the inducibility of the relQ promoter in response to the lack of relP, results in efficient expression of relQ in the relP-deleted background. This positively affects mecA expression and renders the ΔrelP strain highly resistant. These findings indicate an important role for RelQ in the expression of high-level ß-lactam resistance in MRSA.