Rachita Dash

Front Immunol. 2024 Oct 22;15:1442095. doi: 10.3389/fimmu.2024.1442095. eCollection 2024.

ABSTRACT

The mammalian gastrointestinal tract quickly becomes densely populated with foreign microorganisms shortly after birth, thereby establishing a lifelong presence of a microbial community. These commensal gut microbiota serve various functions, such as providing nutrients, processing ingested compounds, maintaining gut homeostasis, and shaping the intestinal structure in the host. Dysbiosis, which is characterized by an imbalance in the microbial community, is closely linked to numerous human ailments and has recently emerged as a key factor in health prognosis. Tuberculosis (TB), a highly contagious and potentially fatal disease, presents a pressing need for improved methods of prevention, diagnosis, and treatment strategies. Thus, we aim to explore the latest developments on how the host's immune defenses, inflammatory responses, metabolic pathways, and nutritional status collectively impact the host's susceptibility to or resilience against Mycobacterium tuberculosis infection. The review addresses how the fluctuations in the gut microbiota not only affect the equilibrium of these physiological processes but also indirectly influence the host's capacity to resist M. tuberculosis. This work highlights the central role of the gut microbiota in the host-microbe interactions and provides novel insights for the advancement of preventative and therapeutic approaches against tuberculosis.

PMID:39502685 | PMC:PMC11534664 | DOI:10.3389/fimmu.2024.1442095

RSC Adv. 2024 Oct 21;14(45):33133-33142. doi: 10.1039/d4ra04945d. eCollection 2024 Oct 17.

ABSTRACT

A method of choice to study the spatio-temporal dynamics of bacterial cell growth and division is to analyze the localization of cell wall synthesis regions by fluorescence microscopy. For this, nascent cell wall biopolymers need to be labeled with fluorescent reporters, like fluorescent d-alanines (FDAs) that can be incorporated into the peptidoglycan. To achieve high spatial and temporal resolution, dense, high-intensity fluorescence labeling must be obtained in the shortest possible time. However, modifications carried by d-Ala can hinder their uptake by the enzymes that incorporate them into the peptidoglycan, such as the d,d-transpeptidases. Conversely, these modifications can impede the elimination of the incorporated d-Ala derivatives by d,d-carboxypeptidases, making the labeling more persistent. In this context, we synthesized clickable d-Alas and tested their incorporation into the peptidoglycan using different labeling approaches, prior or after their conjugation to clickable fluorescent dyes through SPAAC reaction. Our data allow ranking of the d-Ala derivatives in terms of their ease of incorporation and resistance to trimming during one-step, "one-pot" two-step or sequential two-step labeling strategies. We further show that a hybrid "one-step" approach, in which a FDA is used in combination with clickable choline and fluorescent dye, enables two-color co-labeling of peptidoglycan and teichoic acids. Finally, we identify a strategy compatible with the cell fixation required for super-resolution microscopy, by combining one-step labeling with FDA and sequential two-step labeling with clickable choline and fluorescent dye, allowing to obtain two-color high-resolution images of peptidoglycan and teichoic acid synthesis regions.

PMID:39434986 | PMC:PMC11492190 | DOI:10.1039/d4ra04945d

RSC Adv. 2024 Oct 22;14(45):33568-33586. doi: 10.1039/d4ra06117a. eCollection 2024 Oct 17.

ABSTRACT

Antimicrobial resistance (AMR) poses a critical threat to global public health, necessitating the development of novel strategies. AMR occurs when bacteria, viruses, fungi, and parasites evolve to resist antimicrobial drugs, making infections difficult to treat and increasing the risk of disease spread, severe illness, and death. Over 70% of infection-causing microorganisms are estimated to be resistant to one or several antimicrobial drugs. AMR mechanisms include efflux pumps, target modifications (e.g., mutations in penicillin-binding proteins (PBPs), ribosomal subunits, or DNA gyrase), drug hydrolysis by enzymes (e.g., β-lactamase), and membrane alterations that reduce the antibiotic's binding affinity and entry. Microbes also resist antimicrobials through peptidoglycan precursor modification, ribosomal subunit methylation, and alterations in metabolic enzymes. Rapid development of new strategies is essential to curb the spread of AMR and microbial infections. Nanomedicines, with their small size and unique physicochemical properties, offer a promising solution by overcoming drug resistance mechanisms such as reduced drug uptake, increased efflux, biofilm formation, and intracellular bacterial persistence. They enhance the therapeutic efficacy of antimicrobial agents, reduce toxicity, and tackle microbial resistance effectively. Various nanomaterials, including polymeric-based, lipid-based, metal nanoparticles, carbohydrate-derived, nucleic acid-based, and hydrogels, provide efficient solutions for AMR. This review addresses the epidemiology of microbial resistance, outlines key resistance mechanisms, and explores how nanomedicines overcome these barriers. In conclusion, nanomaterials represent a versatile and powerful approach to combating the current antimicrobial crisis.

PMID:39439838 | PMC:PMC11495475 | DOI:10.1039/d4ra06117a

Science. 2024 Nov;386(6721):573-580. doi: 10.1126/science.adn1369. Epub 2024 Oct 31.

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to β-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

PMID:39480932 | DOI:10.1126/science.adn1369

ACS Appl Mater Interfaces. 2024 Nov 13;16(45):61655-61663. doi: 10.1021/acsami.4c13041. Epub 2024 Nov 5.

ABSTRACT

The clinical efficacy of cancer vaccines is closely related to immunoadjuvants that play a crucial role in magnifying and prolonging the immune response. Muramyl dipeptide (MDP), a minimal and conserved peptidoglycan found in almost all bacteria, can trigger robust immune activation by uniquely antagonizing the nucleotide-binding oligomerization domain 2 (NOD2) pathway. However, its effectiveness has been hindered by limited solubility, poor membrane penetration, and rapid clearance from the body. Here, we introduce MDP-presenting polymersomes as artificial nanobacteria (NBA) to boost the antitumor immune response. The NBA, featuring abundant MDP molecules, induces superior stimulation of immune cells including macrophages and bone marrow-derived dendritic cells (BMDCs) compared to free MDP, likely via facilitating immune cell uptake and cooperatively stimulating systemic NOD2 signaling. Importantly, systemic administration of NBA significantly enhances the chemo-immunotherapy of B16-F10 melanoma-bearing mice pretreated with doxorubicin by reversing the immunosuppressive tumor microenvironment. Furthermore, NBA carrying ovalbumin and B16-F10 cell lysates induces robust OVA-IgG antibody production and effectively inhibit tumor growth, respectively. The artificial nanobacteria hold great promise as a potent systemic immunoadjuvant for cancer immunotherapy.

PMID:39498882 | DOI:10.1021/acsami.4c13041

J Am Chem Soc. 2024 Nov 13;146(45):31124-31136. doi: 10.1021/jacs.4c11013. Epub 2024 Oct 30.

ABSTRACT

Stereoselective reduction of dehydroamino acids is a common biosynthetic strategy to introduce d-amino acids into peptidic natural products. The reduction, often observed during the biosynthesis of lanthipeptides, is performed by dedicated dehydroamino acid reductases (dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide, flavin, or F₄₂₀H₂ coenzymes as hydride donors, and little is known about the catalysis performed by the latter family proteins. Here, we perform a bioinformatics-guided identification and large-scale in vitro characterization of five F₄₂₀H₂-dependent dhAARs. We construct an mRNA display-based pipeline for ultrahigh throughput substrate specificity profiling of the enzymes. The pipeline relies on a 4-track selection strategy to deliver large quantities of clean data, which were leveraged to build accurate substrate fitness models. Our results identify a remarkably promiscuous enzyme, referred to as MaeJ_C, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition requirements. We integrate MaeJ_C into a thiopeptide biosynthetic pathway to produce d-amino acids-containing thiopeptides, demonstrating the utility of MaeJ_C for the programmable installation of d-amino acids in ribosomal peptides.

PMID:39474650 | DOI:10.1021/jacs.4c11013

Chem Biomed Imaging. 2023 Dec 31;2(2):98-116. doi: 10.1021/cbmi.3c00117. eCollection 2024 Feb 26.

ABSTRACT

With high efficiency, mild conditions, and rapid reaction rate, click reactions have garnered much attention in the field of bioimaging since proposed by Sharpless et al. in 2001 (Angew. Chem., Int. Ed.2001, 40, 2004-2021). Inspired by the regenerative pathway of d-luciferin in fireflies, Liang et al. (Nat. Chem.2010, 2, 54-60) raised a 2-cyanobenzothiazole (CBT)-cysteine (Cys) click condensation reaction in 2010, which exhibits a higher second-order reaction rate (9.19 M^-1 s^-1) and superior biocompatibility. As it has been developed in the past decade, remarkable progress has been made in the construction of enzyme-instructed CBT-Cys-based bioimaging probes. This review introduces the concept of the CBT-Cys click reaction, elucidates the mechanism of the CBT-Cys click reaction, and concerns the development progress of CBT-Cys reaction and its derived reactions [i.e., 2-cyano-6-hydroxyquinoline (CHQ)-Cys reaction and 2-pyrimidinecarbonitrile (PMN)-Cys reaction]. Furthermore, we give a comprehensive and up-to-date review of enzyme-instructed CBT-Cys-like click reaction-based probes with significantly enhanced imaging signal and contrast for various bioimaging modes, including fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, and positron emission tomography. In the end, we discuss the possible challenges and opportunities that may arise in the future.

PMID:39474483 | PMC:PMC11504589 | DOI:10.1021/cbmi.3c00117

J Am Chem Soc. 2024 Nov 13;146(45):31124-31136. doi: 10.1021/jacs.4c11013. Epub 2024 Oct 30.

ABSTRACT

Stereoselective reduction of dehydroamino acids is a common biosynthetic strategy to introduce d-amino acids into peptidic natural products. The reduction, often observed during the biosynthesis of lanthipeptides, is performed by dedicated dehydroamino acid reductases (dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide, flavin, or F₄₂₀H₂ coenzymes as hydride donors, and little is known about the catalysis performed by the latter family proteins. Here, we perform a bioinformatics-guided identification and large-scale in vitro characterization of five F₄₂₀H₂-dependent dhAARs. We construct an mRNA display-based pipeline for ultrahigh throughput substrate specificity profiling of the enzymes. The pipeline relies on a 4-track selection strategy to deliver large quantities of clean data, which were leveraged to build accurate substrate fitness models. Our results identify a remarkably promiscuous enzyme, referred to as MaeJ_C, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition requirements. We integrate MaeJ_C into a thiopeptide biosynthetic pathway to produce d-amino acids-containing thiopeptides, demonstrating the utility of MaeJ_C for the programmable installation of d-amino acids in ribosomal peptides.

PMID:39474650 | DOI:10.1021/jacs.4c11013

J Med Chem. 2024 Nov 14;67(21):19612-19622. doi: 10.1021/acs.jmedchem.4c01956. Epub 2024 Oct 25.

ABSTRACT

Hydrocarbon-determined shake-flask measurements have demonstrated great utility for optimizing lipophilicity during early drug discovery. Alternatively, chromatographic methods confer reduced experimental error and improved handling of complex mixtures. In this study, we developed a chromatographic approach for estimating hydrocarbon-water shake-flask partition coefficients for a variety of macrocyclic peptides and other bRo5 molecules including PROTACs. The model accurately predicts experimental shake-flask measurements with high reproducibility across a wide range of lipophilicities. The chromatographic retention times revealed subtle conformational effects and correlated with the ability to sequester hydrogen bond donors in low dielectric media. Estimations of shake-flask lipophilicity from our model also accurately predicted trends in MDCK passive cell permeability for a variety of thioether-cyclized decapeptides. This method provides a convenient, high-throughput approach for measuring lipophilic permeability efficiency and predicting passive cell permeability in bRo5 compounds that is suitable for multiplexing pure compounds or investigating the properties of complex library mixtures.

PMID:39453819 | PMC:PMC11571107 | DOI:10.1021/acs.jmedchem.4c01956

Chem Rev. 2024 Nov 13;124(21):12213-12241. doi: 10.1021/acs.chemrev.4c00422. Epub 2024 Oct 25.

ABSTRACT

Technological advances and breakthrough developments in the pharmaceutical field are knocking at the door of the "undruggable" fortress with increasing insistence. Notably, the 21st century has seen the emergence of macrocyclic compounds, among which cyclic peptides are of particular interest. This new class of potential drug candidates occupies the vast chemical space between classic small-molecule drugs and larger protein-based therapeutics, such as antibodies. As research advances toward clinical targets that have long been considered inaccessible, macrocyclic peptides are well-suited to tackle these challenges in a post-rule of 5 pharmaceutical landscape. Facilitating their discovery is an arsenal of high-throughput screening methods that exploit massive randomized libraries of genetically encoded compounds. These techniques benefit from the incorporation of non-natural moieties, such as non- proteinogenic amino acids or stabilizing hydrocarbon staples. Exploiting these features for the strategic architectural design of macrocyclic peptides has the potential to tackle challenging targets such as protein-protein interactions, which have long resisted research efforts. This Review summarizes the basic principles and recent developments of the main high-throughput techniques for the discovery of macrocyclic peptides and focuses on their specific deployment for targeting undruggable space. A particular focus is placed on the development of new design guidelines and principles for the cyclization and structural stabilization of cyclic peptides and the resulting success stories achieved against well-known inaccessible drug targets.

PMID:39451037 | PMC:PMC11565579 | DOI:10.1021/acs.chemrev.4c00422

RSC Adv. 2024 Oct 21;14(45):33133-33142. doi: 10.1039/d4ra04945d. eCollection 2024 Oct 17.

ABSTRACT

A method of choice to study the spatio-temporal dynamics of bacterial cell growth and division is to analyze the localization of cell wall synthesis regions by fluorescence microscopy. For this, nascent cell wall biopolymers need to be labeled with fluorescent reporters, like fluorescent d-alanines (FDAs) that can be incorporated into the peptidoglycan. To achieve high spatial and temporal resolution, dense, high-intensity fluorescence labeling must be obtained in the shortest possible time. However, modifications carried by d-Ala can hinder their uptake by the enzymes that incorporate them into the peptidoglycan, such as the d,d-transpeptidases. Conversely, these modifications can impede the elimination of the incorporated d-Ala derivatives by d,d-carboxypeptidases, making the labeling more persistent. In this context, we synthesized clickable d-Alas and tested their incorporation into the peptidoglycan using different labeling approaches, prior or after their conjugation to clickable fluorescent dyes through SPAAC reaction. Our data allow ranking of the d-Ala derivatives in terms of their ease of incorporation and resistance to trimming during one-step, "one-pot" two-step or sequential two-step labeling strategies. We further show that a hybrid "one-step" approach, in which a FDA is used in combination with clickable choline and fluorescent dye, enables two-color co-labeling of peptidoglycan and teichoic acids. Finally, we identify a strategy compatible with the cell fixation required for super-resolution microscopy, by combining one-step labeling with FDA and sequential two-step labeling with clickable choline and fluorescent dye, allowing to obtain two-color high-resolution images of peptidoglycan and teichoic acid synthesis regions.

PMID:39434986 | PMC:PMC11492190 | DOI:10.1039/d4ra04945d

J Am Chem Soc. 2024 Oct 30;146(43):29429-29440. doi: 10.1021/jacs.4c08520. Epub 2024 Oct 21.

ABSTRACT

There is a demand for site-selective peptide/protein conjugation chemistry that is fully reversible in a stimulus-responsive manner. The contemporary methods for site-selective protein modification enable the preparation of homogeneous protein-small molecule conjugates, which are indispensable for drug delivery and chemical biology purposes, but such chemistries are usually irreversible. In contrast, the existing reversible protein labeling techniques are generally not site-selective. Here, we report an mRNA display-enabled de novo discovery of a pair of peptides which selectively react with each other to form a conjugate that is stable under neutral conditions (pH 7.5) but rapidly dissociates into the constituents at pH 10. A Cys thiol of peptide CP1 rapidly reacts (k₁ = 340 M^-1·s^-1) with the isothiocyanate moiety in partner ITC6 to furnish a stable dithiocarbamate (t_1/2 = 6 h at pH 7.5). We show that the pH-responsive nature of the reaction (conjugate's t_1/2 = 5 min at pH 10) can be leveraged to utilize ITC6 (1) as a pull-down handle to selectively isolate CP1 from cell lysates and (2) as a temporary protecting group to protect CP1 from nonspecific Cys labeling reagents such as iodoacetamide. Altogether, the chemistry developed here complements the existing approaches and is applicable in a variety of chemical biology settings where selective reversible reactions are needed.

PMID:39432830 | DOI:10.1021/jacs.4c08520