Antimicrobial Resistance

Area Co-Ordinators

  • Dr. B. N. Singh

    Antimicrobial Resistance

  • Dr. Gautam Panda

    Antimicrobial Resistance

Vision and Goal:

The World Health Organization(WHO) has defined Antimicrobial resistance (AMR) as the ability of a microorganism to stop an antimicrobial from working against it. As a result, standard treatments become ineffective, infections persist and may spread to others. It has further stated “AMR is of particular concern in developing nations, including India, where the burden of infectious disease is high and healthcare spending is low. The country has among the highest bacterial disease burden in the world. Antibiotics, therefore, have a critical role in limiting morbidity and mortality in the country.” WHO in the 2017 report categorically states that tuberculosis and some gram-negative infections caused by ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens fall in the highest critical priority category and have to be treated with utmost urgency.
The global objectives of CSIR-CDRI program:
▶ Drug Discovery studies against drug-resistant mycobacterial infections and ESKAPE pathogens
▶Discovery of new therapeutic strategies/interventions/diagnostics by Advancing Knowledge Frontiers

Design and synthesis

Natural product tryptanthrin derivatives as new antitubercular agents

Our research interest involves the design of novel molecules based on biologically active natural products. Since the natural product tryptanthrin and its derivatives were exemplified with potent in vitro activity against Mycobacterium tuberculosis (Mtb), we have designed new derivatives of tryptanthrin for the synthesis and biological evaluation. Currently, novel methods have been designed and optimization conditions are in progress.

Synthesis of unnatural antimicrobial peptide hybrids

Chemically synthesized linear antimicrobial peptides are limited by their low biostability, high clearance, poor membrane permeability and solubility and the difficulty of being orally administered. To improve bioavailability of antimicrobial peptides, we proposed and synthesized various structurally modified peptides by incorporating D-amino acids, N-alkylation and fused heterocycles. Several compounds have been synthesized and are being evaluated. Chemical diversity around diaryl methyl amines Several compounds of the family were synthesized and evaluated for anti-bacterial activity including selected ESKAPE pathogens. Initially promising leads are being investigated further.

Trisubstituted methanes, naphthyridines and quinoline containing compounds as antitubercular agents

Under this heading, the following compounds were designed and synthesized. 13 compounds were evaluated and their cytotoxicity is under evaluation.

Corannulene derived hydrogelators as possible Cationic Antimicrobial Peptides (CAPs)

The combination of promptly functionalized amine and carboxyl groups stick to a chiral central core along with one potentially diverse molecule having bowl shape and greater hydrophobicity provides a unique three-dimensional structure with high attention. Apart from amino acids, no other readily available building block contains two orthogonal functional groups that can be modified by convenient chemistry. In this endeavor we have for the first time designed and synthesized four novel kinds of corannulene containing unnatural acids and their peptides

Thiocyanated oxazoline and thiazoline derivatives

The synthetic routes for accessing thiocyanated oxazoline and thiazoline derivatives have been developed and proposed structures for further study are based on the schematics depicted below:

Biological screening

Assay summary of compounds screened against Mycobacterium tuberculosis

Compounds with activity are being further optimized to improve efficacy.

Assay summary of compounds screened against ESKAPE pathogens

Compounds with activity are being further optimized to improve efficacy.

Team Members:

Above Left to Right : Dr A.A. Sahasrabuddhe, Dr Malleswara Rao Kuram, Dr Mukesh Pashupaleti, Dr Ravindra Kumar, Dr Ajay Kumar Srivastava, Dr PK Shukla, Dr Siddharth Chopra, Dr Tejender S Thakur, Dr S.K. Singh, Dr Kalyan Mitra

Below Left to Right : Dr Y.K. Manju, Dr Nayan Ghosh, Dr Gautam Panda, Dr M.I. Siddiqi, Dr R Ravishankar, Dr Atul Kumar, Dr B.N. Singh, Dr K.K. Srivastava, Dr Vinita Chaturvedi

Core Competencies and Activities:

The AMR team uses several cutting edge drug discovery platform technologies involving screening, molecular & structural biology, chemistry, computational biology and allied areas. The team has characterized several novel targets and has identified several new scaffolds through early target discovery and research that feeds into the drug discovery pipeline of the institute.

Advancing Knowledge Frontiers

Mycobacterium bovis sigF mutant exhibits altered surface phenotype and compromised pathogenesis

Sigma factor F (SigF) is an alternate sigma factor, widely conserved in pathogenic and non-pathogenic mycobacteria, suggesting its larger role in addition to regulation of virulence genes. We generated a ΔsigF mutant in M. bovis which displayed distinct colony morphotype suggestive of deficiency in surface properties (Fig 2A and 2B). The loss in phenotype was restored in the complemented ΔsigF mutant (Fig 2A and 2B). The ΔsigF mutant showed marked depletion in key cord forming lipids, trehalose 6-6’-dimycolate, trehalose 6-monomycolate and phthiocerol dimycocerosate (Fig 3B and 3E). Comparative proteomics revealed diminished level of several proteins predicted to have roles in cell surface properties, stress response and virulence associated functions. Proteome analysis of M. bovis biofilms highlighted the role of SigF regulated proteins in biofilm formation as several of them appeared at lower levels in the ΔsigF mutant. One of them was a key metabolic enzyme, malate synthase G (Mb1868c). Consistent with its pleiotropic role, the diminished level of Mb1868c in the ΔsigF mutant resulted in reduced adherence of the mutant bacilli to lung epithelial cells. In summary, we report novel morphotypes of the M. bovis ΔsigF mutant and provide rationale for their in vitro and in vivo phenotypes, which improve our understanding of the role of SigF in mycobacterial biology.

Fig. Colony morphotypes of M. bovis wild-type (Mb), ΔsigF mutant (Mb10) and complemented strain (Mb11). (A) The ΔsigF mutant colonies appear smooth. (B) Appearance of M. bovis strains on LJ slants. Accumulation of Nile red 2 µM (C) and EtBr 6 µM (D) by M. bovis wild-type (Mb), ∆sigF mutant (Mb10) and complemented strain (Mb11) were measured by fluorescence spectroscopy. The data are mean ± SD from three independent experiments (**** p <0.0001).

Fig. 2D-TLC analysis. Nonpolar lipids from M. bovis wild-type (A), ΔsigF mutant (B) and complemented strain (C). Polar lipids from M. bovis wild-type (D), ΔsigF mutant (E) and complemented strain (F). The arrows indicate the differential lipids content. PDIM, TMM and TDM refer to Phthiocerol dimycocerosate, Trehalose monomycolate and Trehalose dimycolate, respectively. (G) Shows the quantitation of the PDIM levels in different strains using image analysis software (GE Healthcare). The data were normalized to those for the wild-type strain and represent mean ± SD of three independent experiments (*** p <0.001). (H) DNase treatment of M. bovis biofilms. M. bovis wild-type biofilms were incubated with PBS, PBS and buffer (reaction buffer), PBS + DNase, heat inactivated (HI) DNase and buffer + DNase. Biofilms were quantified via crystal violet staining. Data represent mean ± SD of three independent experiments (*** p <0.001).

Development of novel antimycobacterial peptides using database filtering and three-dimensional modeling approach

The cell wall of Mycobacterium tuberculosis (Mtb) provides a major defensive mechanism from antibiotics, phagocytic vesicles in macrophages and d esiccation in dry sputum. Host defense peptides (HDPs), our "natural antibiotics", are major component of innate immunity and evolutionary conserved, host defense molecules, having a direct action on microbial membranes with a low risk of resistance development. The peptides which have potential to assume helical structure in liquid cultures do have antimicrobial activity. Also, the moderately short cationic and hydrophobic peptides derived from the conserved domains of various proteins have antimicrobial activity under physiological conditions without any toxicity issues. However, the composition of the mycobacterial membrane differs significantly from that of bacteria, and it cannot be concluded that all AMPs, which have been identified as pore/ lesion-forming HDPs against bacteria, also induce lesions in mycobacteria. We predicted that the helical domains of Glutathione s-transferase, (GST)-theta might have antimycobacterial activity or could give us templates for further optimization. So in this work, we designed potent HDPs than those found in nature, to construct a random in silico peptide library. Then by using “database filtering” and three-dimensional (3D) modeling approach peptide sequences were selected for the synthesis, followed by in vitro analysis. We explored peptides derived from the GST for antimycobacterial activity. The candidate peptides, shown in wheel diagram representations (Fig 4), were developed. The helical wheel diagrams demonstrate the amphipathic nature of peptides and in order to see the effect of peptides on membranes, we did a scanning electron microscopy study with peptides at 2X MIC concentrations (Fig. 5).

The results show that all the tested peptides induced cytoplasmic condensation and cell wall modification (thinning and budding) (YRA-15 and YWW-15) or destruction (RWW-15 and YQA-15). The cytotoxicity studies indicated that the peptides were not toxic to the human cells. In the optimized series, peptide with higher charge but lower hydrophobicity, showed signs of toxicity at the lowest concentration (50 µM), whereas the peptides with lower charge and higher hydrophobicity showed lower toxicity (Fig 5). Among all the peptides four peptides showed significant gain in the activity. This study indicates that peptide to be active against Mycobacterium tuberculosis H37Ra should have a charge of +4 and hydrophobicity > 60% and less than 70%.

Fig. Scanning electron microscopy images showing the mode of action of the peptides (upper) and cytotoxicity profile (lower) of hit molecules.

Identification of fragment inhibitors of Mycobacterium tuberculosis NAD+-dependent DNA Ligase A that target ‘serial-remodeling’

The NAD+-dependent DNA ligase (LigA) is a multi-domain, essential, bacterial enzyme that functions as the principal replicative ligase. Differences between the human ATP-dependent DNA Ligase-I (Lig1) and LigA makes the latter an attractive antibacterial target. We solved co-crystal structures with NMN, AMP and inhibitors identified from fragment-screening. Inhibitors reported previously mainly compete with AMP and bind to the 1B sub-domain of the adenylation domain. We recently screened a fragment library against the NMN-binding pocket and identified 2 fragments that inhibit LigA activity with IC50 in the low µM range. Experiments involving E. coli GR501 strain that harbors its own temperature-sensitive LigA, rescued with Mtb LigA, show that the fragments inhibit bacterial growth by targeting Mtb LigA in vivo. Additionally, the experiments showed that the inhibitors can distinguish between NAD+- and ATP-dependent ligases. The NMN binding pocket on subdomain 1A has been shown to be a druggable target site for developing anti-LigA therapeutic strategies.

Nef regulates protein trafficking in HIV Pathogenesis

HIV-1 Nef regulates several cellular functions in an infected cell. The molecular mechanism(s) underlying Nef-dependent cellular function(s) are unable to define how the events are coordinately regulated in the host cell. In this study, our laboratory deciphered the role of Nef on Rab GTPases - dependent complex vesicular trafficking. Expression profiling of Rabs in Nef-expressing cells showed that Nef differentially regulates the expression of individual Rabs in a cell-specific manner. Further analysis of Rabs in HIV-1NL4-3 or ΔNef infected cells demonstrated that Nef protein was responsible for variation in Rabs expression. Using a panel of competitive peptide inhibitors against Nef, we identified the critical domain of HIV-1 Nef involved in modulation of Rabs expression. The molecular function of Nef-mediated up-regulation of Rab5 and Rab7 and down-regulation of Rab11 increased the transport of SERINC5 from the cell surface to lysosomal compartment. Moreover, Nef-dependent increase in Rab27 expression assists its own release via exosomes. Reversal of Rabs expression using competitive inhibitors against Nef reduced viral release and infectivity of progeny virions. Overall, this study uncovers a new paradigm that Nef differentially regulate the expression of Rabs protein in infected cells to hijack the host intracellular trafficking, which augment viral replication and HIV-1 pathogenesis.

A long-term lipid only diet can convert Mycobacterium tuberculosis to hard-to-grow persisters

The differentially detectable (DD) persisters, of Mycobacterium tuberculosis (Mtb), are persisters that fail to form colonies on agar media when de-stressed. Since in the host, Mtb primarily survives by utilizing lipids, we used a long-term lipid diet model to induce DD persisters of Mtb. Persisters were induced by replacing the dextrose-containing medium with one containing fatty acids (FAM). After 2, 4 or 6 weeks, CFU and most probable number assays were performed; the difference between the two gave an estimate of DD persisters (Fig. 7). Since rifampicin has been shown to induce formation of DD persisters in vitro, one set of FAM cultures were also given short- term rifampicin stress after 2, 4 or 6 weeks.

Fig. The fraction of DD persisters increased with time and rifampicin treatment enhanced the effect of fatty acids, at 2 and 4 weeks. At six weeks, even in the absence of rifampicin, approximately 95 % cells were DD persisters. The DD persisters (Fig. panel B) were found to be more drug tolerant than the colony forming persisters (Fig. panel A) under the similar culture conditions. The DD persisters were vulnerable to drugs interfering with bacterial respiration such as thioridazine (TRZ), bedaquiline and clofazimine (Figure), but were tolerant to rifampicin and moxifloxacin. The study indicates potential formation of DD persisters of Mtb in a lipid-rich microenvironment in the host even before antibiotic therapy.