Ananya Naick

J Chem Inf Model. 2026 Feb 23;66(4):1982-2005. doi: 10.1021/acs.jcim.5c02731. Epub 2026 Feb 9.

ABSTRACT

Biological membranes are crucial for cellular integrity and function, but their selective permeability can be compromised by various peptides and proteins, such as antimicrobial peptides (AMPs) and pore-forming proteins/toxins (PFPs/PFTs). These molecules induce membrane permeabilization through diverse mechanisms, ranging from the formation of well-defined pores to more nuanced disruptions of the lipid bilayer. Understanding molecular mechanisms underlying membrane integrity disruption is vital for developing novel tools to be applied in medicine, biotechnology, and agriculture. However, due to their transient and dynamic nature, characterizing membrane-disrupting mechanisms is a significant experimental challenge. In silico methods, particularly all-atom and coarse-grained molecular dynamics (MD) simulations, are an indispensable tool to complement and enrich experimental studies, and can offer detailed insights into peptide/protein-membrane interactions, insertion, oligomerization, and pore formation. This review provides a comprehensive overview of the structural and mechanistic diversity of AMPs and PFPs, highlighting representative case studies and discussing key challenges emerging from MD simulations.

PMID:41662490 | DOI:10.1021/acs.jcim.5c02731

Microbiology (Reading). 2026 Feb;172(2):001657. doi: 10.1099/mic.0.001657.

ABSTRACT

Cationic peptides, particularly those rich in arginine and/or lysine residues, are usually promising antimicrobial agents effective at low concentrations in laboratory settings. However, their applicability in pharmaceutics and biotechnology is currently limited due to their susceptibility to biological enzymatic processes and (in some cases) toxicity to host cells. To address this, we screened eight linear arginine-rich peptides for their haemolytic properties and antimicrobial activity using a set of computational and experimental assays. Inspired by our previous results on R4F4, we then designed three modified peptides based on an R4F4 backbone, R4F4-C16, D-R4F4 and cyclic R4F4, and one based on R4 (R4-C16). Amongst the tested linear peptides containing only natural amino acids, R4F4 exhibited the strongest antibacterial activity; however, this effect was reduced in the presence of human serum and trypsin. Conversely, our study demonstrated that cyclization and substitution to its d-amino acid enantiomer significantly enhanced stability and activity of R4F4, whilst in the presence of proteases. As revealed by fluorescence imaging, microscopy RNA sequencing analysis, the mode of action involves complex and dynamic events. This multifaceted antimicrobial mechanism integrates alterations in membrane permeability, modulation of intracellular reactive oxygen species levels and changes in transcriptomic signature profiles. At the molecular level, notable changes were observed in the bacterial expression of genes associated with metabolic pathways and biological processes. Furthermore, R4F4-derived peptides showed substantial antibiofilm activity in preventing the formation and disruption of mature biofilms, together with good cytocompatibility, highlighting the potential for clinical applicability. In conclusion, this study emphasizes the importance of optimizing the stability of peptide-based antimicrobials, particularly those rich in arginine, and highlights the advantages of incorporating d-amino acids and cyclization for enhanced performance. This information will prove useful in the future design of antimicrobial peptides. In addition, the molecular perspective on peptide-induced gene expression changes, as identified by RNA-seq, broadens our understanding of antimicrobial peptides' activities and provides a clearer picture of their versatile mechanisms.

PMID:41637123 | PMC:PMC12873548 | DOI:10.1099/mic.0.001657

Biochim Biophys Acta Biomembr. 2026 Jan 13;1868(2):184499. doi: 10.1016/j.bbamem.2026.184499. Online ahead of print.

ABSTRACT

Antimicrobial peptides (AMPs) represent a current strategy to develop new antibiotics against multi-resistant pathogens. The potential antibiotic activity of AMPs is related to their amphipathic properties and the presence of positively charged residues, which may interact with the negatively charged bacterial membranes. In contrast, they exhibit lower interaction with the eukaryotic, neutrally charged membranes. This is the primary reason AMPs can distinguish between eukaryotic and prokaryotic membranes. AMPs are usually modified or designed de novo, and their properties can be changed by inserting specific amino acid residues into their sequence. To assist in the rational design of AMPs, it is helpful to explore the biophysical changes they may induce in target cell membranes. Therefore, bacterial and eukaryotic model lipid membranes have been extensively used for this purpose. Parameters such as selective binding, lipid membrane interactions, membrane packing, permeability, hydration, and restructuring facilitate the exploration of peptide regions of interest. These parameters can be studied using various physicochemical techniques, including differential scanning calorimetry, X-ray diffraction, nuclear magnetic resonance, and fluorescence spectroscopy. This review aims to provide a practical guide to the main biophysical techniques used to explore the potential antibiotic activity of AMPs using model membranes, and to examine lipid-peptide interactions in order to define the mechanisms of action of these antimicrobial peptides. These techniques determine whether the peptide interacts specifically with bacterial membranes, the preferred bacterial target of a given AMP, the binding affinities of AMPs, potential pore formation and its geometry, and the impact of these interactions on both bacterial and eukaryotic membranes.

PMID:41539421 | DOI:10.1016/j.bbamem.2026.184499

ACS Infect Dis. 2026 Jan 9. doi: 10.1021/acsinfecdis.5c00833. Online ahead of print.

ABSTRACT

The natural product fidaxomicin (Fdx) is a narrow-spectrum antibiotic clinically prescribed for the treatment of Clostrodioides difficile infections. However, limited cellular uptake reduces its therapeutic potential, particularly against Gram-negative bacteria and mycobacteria. In this study, we investigated Thiol-Mediated Uptake (TMU) to promote the delivery of Fdx into bacterial cells. We synthesized a library of Fdx derivatives bearing cyclic dichalcogenide moieties and evaluated their antimicrobial properties against C. difficile and Mycobacterium tuberculosis, respectively. Remarkably, the synthetic Fdx derivatives retained strong levels of antibacterial activity, and the disulfide-containing analogs outperformed their all-carbon control counterparts in many instances. We then developed a systematic study to investigate the mechanistic impact of the introduced disulfide functionalities by conducting experiments with TMU inhibitors and quantifying intracellular accumulation in Mycobacterium bovis BCG, a model organism for M. tuberculosis, via LC-MS/MS. While complete disentanglement of the factors influencing activity was not feasible, features such as compound stability and lipophilicity were identified as significant contributors. Overall, the superior performance of disulfide analogs suggests that differences in cellular entry or intracellular processing, potentially related to TMU, are involved. This work highlights that TMU remains a viable approach for modulating the uptake of therapeutic agents into bacterial cells.

PMID:41510831 | DOI:10.1021/acsinfecdis.5c00833

Mini Rev Med Chem. 2026 Jan 7. doi: 10.2174/0113895575404841251121074713. Online ahead of print.

ABSTRACT

Antimicrobial peptides are tiny molecular polypeptides that help living things fight off foreign microbes. According to recent studies, they come from a wide range of sources and can be found in a wide range of creatures, including microbes, plants, and animals. The method of action has the ability to act on intracellular targets in addition to the traditional membrane permeability mechanism. In terms of technology, antimicrobial peptides offer special benefits. They may successfully suppress a range of bacteria in the antibacterial area and are anticipated to address the issue of drug-resistant bacteria. Antiviral medications, such as those that block the herpes virus or influenza, are predicted to become a new generation of antiviral medications when they can precisely target cancer cells without harming healthy cells. This holds enormous promise for the field of biological medicine. However, it also faces problems such as production, safety, and activity retention, which limit its further development and large-scale application. The introduction of non-natural amino acids can address this issue because the natural antibacterial peptide is easily degraded by the protease. In addition, by introducing non-natural amino acids with special structures and properties, not only can the interaction between AMP and a target be optimized, but also the functional range of AMP can be expanded, and diversified innovation of functions is realized. This study primarily examined the use of non-natural amino acids in the creation of antimicrobial peptides. By exploiting the unique properties of these amino acids, we address current limitations of antimicrobial peptides and provide theoretical guidance for their further development and broad application.

PMID:41510715 | DOI:10.2174/0113895575404841251121074713

Bioconjug Chem. 2025 Dec 31. doi: 10.1021/acs.bioconjchem.5c00541. Online ahead of print.

ABSTRACT

Small-molecule immune modulators offer a promising alternative to biologics, such as antibodies, for cancer immunotherapy. A key example is the TLR7 agonist imiquimod (IMQ), which has already been approved for the treatment of various dermatological malignancies. Nevertheless, the clinical use of IMQ is limited to tumors amenable to topical application, as its systemic administration poses a high risk of severe inflammatory toxicity due to the widespread expression of TLRs. Therefore, to extend the use of TLR7 agonists to the treatment of other solid tumor types, we developed a β-glucuronidase-responsive albumin-binding prodrug designed for the selective delivery of IMQ within the tumor microenvironment. This prodrug masks IMQ's immunogenicity, allowing for its administration in immunocompetent mice without eliciting the systemic side effects associated with TLR7 agonists. However, the β-glucuronidase-catalyzed prodrug activation enables the selective, tumor site-specific release of IMQ, thereby restoring its biological activities. This controlled delivery promotes M1 macrophage polarization, T cell activation, and an increase in IgG levels exclusively within malignant tissues without affecting the healthy organs that are sensitive to TLR7 agonists. This study demonstrates that targeting tumor microenvironment specificities represents a promising approach for developing selective cancer immunotherapies based on small-molecule immune modulators.

PMID:41472650 | DOI:10.1021/acs.bioconjchem.5c00541

Bioconjug Chem. 2026 Jan 7. doi: 10.1021/acs.bioconjchem.5c00461. Online ahead of print.

ABSTRACT

The preparation of antibody drug conjugates (ADC) most often relies on a linear sequence to elaborate the small molecule component, followed by a final bioconjugation step to attach it to its immunoglobulin partner. This linear and iterative approach is incompatible with expedient parallel synthesis and process automation. Here, we describe the design and implementation of a general modular platform for the assembly of ADCs that enables facile variation in the nature of the payload, the linker composition, and the type of bioconjugation technique used. A library of antibody conjugates bringing together several different antibodies and payloads was prepared in a convergent fashion using a range of conjugation methods, as well as cleavable or noncleavable linker technology. Aside from offering a direct comparison of different conjugation method performances, this approach enables a more targeted optimization strategy of conjugate properties by deconvoluting bioconjugation and payload attachment.

PMID:41496718 | DOI:10.1021/acs.bioconjchem.5c00461

Bioconjug Chem. 2026 Jan 21;37(1):160-168. doi: 10.1021/acs.bioconjchem.5c00554. Epub 2026 Jan 10.

ABSTRACT

A fluorinated, disulfide-cross-linked polyplex platform (PFND) was developed for safe and potent cytosolic delivery of siRNA. Branched PEI (25 kDa) was first perfluoro-acylated to yield a membrane-zipper PF backbone, followed by orthogonal installation of azide (PF-N₃) and strained alkyne (PF-DBCO) handles that undergo in situ copper-free click cross-linking in the presence of siRNA. The resulting 60 nm polyplexes (PDI < 0.1, ζ potential of approximately +22 mV) are stable in 10 mg mL^-1 heparin (<5% siRNA leakage) yet quantitatively disassemble within 60 min in 10 mM glutathione, liberating the siRNA payloads. Compared with the commercial gold standard of Lipofectamine 3000, PFND delivers 2- to 3-fold more Cy5-siRNA into HeLa, HepG2, and MDA-MB-468 cells without detectable hemolysis or cytotoxicity. Consequently, 10 nM siGAPDH delivered by PFND silences approximately 93-98% of GAPDH mRNA across the three lines, remarkably outperforming gold-standard transfection reagents. The proposed reversible "locked-outside/labile-inside" design reconciles extracellular stability with rapid intracellular release, offering a valid tool for utilities of high-throughput siRNA screening or subject to be developed further for potential clinical translation of RNAi-based therapeutics.

PMID:41518301 | DOI:10.1021/acs.bioconjchem.5c00554

J Org Chem. 2026 Jan 23;91(3):1257-1263. doi: 10.1021/acs.joc.5c00995. Epub 2026 Jan 7.

ABSTRACT

We report a new method for the synthesis of N-benzo[d]thiazol-2-sulfonyl (Bts, betsyl)-protected amino acids, yielding building blocks amenable to solid-phase peptide synthesis (SPPS) in one step from their corresponding free amino acids. The procedure uses sodium benzo[d]-thiazol-2-sulfinate (sodium betsylate) and buffered aqueous oxidative conditions to generate unstable benzo[d]-thiazol-2-sulfonyl chloride (Bts-Cl) in situ. This epimerization-free method was used to generate a library of betsylated amino acids containing various protected and unprotected side chains. Further utility was demonstrated by incorporating an N-terminal betsylated residue on a linear pentapeptide as a means of preparing a macrocycle via an on-resin Mitsunobu reaction. Additionally, betsylated amino acids proved to be suitable for peptide synthesis in solution with newer coupling reagents such as COMU and TCFH/NMI. Deprotection can be performed under basic or reductive conditions, allowing for orthogonality with other commonly used protecting groups.

PMID:41502210 | DOI:10.1021/acs.joc.5c00995

ACS Synth Biol. 2025 Dec 26. doi: 10.1021/acssynbio.5c00613. Online ahead of print.

ABSTRACT

Cyanobactin biosynthetic pathways are used in synthetic biology approaches to create large, peptide-based chemical libraries with drug-like features such as N-C macrocyclization and prenylation. It remains challenging to express enzymes from multiple RiPP pathways to rationally produce the desired products. Here, we designed a simple yet robust method aimed to produce and assess multiple enzymes, fusing biosynthetic genes together in a well-expressed, soluble construct that enables production of macrocyclic peptides and selectively appends C₅, C₁₀, or C₁₅ isoprenoids to tyrosine side chains. A library was developed and assayed, defining the sequence features necessary for prenylation and providing an overall >40% success rate of using a library with an estimated maximum size of 2.6 million peptide derivatives. This flexible and robust system enables the generation of novel compounds and libraries of such compounds with minimal side products in living organisms.

PMID:41451912 | DOI:10.1021/acssynbio.5c00613

bioRxiv [Preprint]. 2025 Oct 31:2025.10.30.685531. doi: 10.1101/2025.10.30.685531.

ABSTRACT

The blood-brain barrier (BBB) is an indispensable, selectively permeable interface that controls the entry and exit of nutrients, ions and waste products into the brain. Despite its biological importance, most measurements of BBB permeability rely on dyes that suffer from nonspecific signals, lack of spatial fidelity, and incompatibility with longitudinal or repeated measurements. Here we present HaloTrace: a method which leverages the HaloTag ligand-receptor tool to generate a precise spatiotemporal readout of BBB integrity that avoids major pitfalls of existing methods. We present evidence that the fluorescent HaloTag ligand has minimal interactions with blood contents but can enter the brain specifically at sites of BBB dysfunction, where it covalently binds to nearby HaloTag receptors. The ligand accumulates in the brain during its short lifetime in circulation and is stably anchored in place for at least 24 hours. Unlike existing tracers, free ligand is not retained in the blood vessels at detectable levels, so the entirety of ligand fluorescence represents true BBB leakage. Furthermore, we demonstrate that HaloTrace can quantify BBB permeability at multiple discrete timepoints prior to the experiment endpoint. This offers researchers the ability to study the progression or resolution of BBB permeability in a way current methods cannot. HaloTrace is thus uniquely poised to characterize the spatiotemporal dynamics of BBB leakage in mouse models.

PMID:41279380 | PMC:PMC12636304 | DOI:10.1101/2025.10.30.685531

J Med Chem. 2025 Dec 11. doi: 10.1021/acs.jmedchem.5c02537. Online ahead of print.

ABSTRACT

Complement component 5 (C5) is a protein in the complement cascade and a part of the innate immune system that has been clinically validated as a therapeutic target for several immune-mediated diseases including generalized myasthenia gravis (gMG). In this paper, we discuss the early discovery of zilucoplan, a macrocyclic peptide drug, which was identified via innovative extreme diversity mRNA display (Ma, Z.; Hartman, M. C. T. In Vitro Selection of Unnatural Cyclic Peptide Libraries via mRNA Display. In Ribosome Display and Related Technologies: Methods and Protocols; Douthwaite, J. A., Jackson, R. H., Eds.; Springer: New York, 2012; pp 367-390.) against C5 and approved for the treatment of gMG. We highlight the key steps and rationale behind the peptide medicinal chemistry optimization of the initial screening hits, that led to improved potency, stability, and pharmacokinetic properties.

PMID:41379101 | DOI:10.1021/acs.jmedchem.5c02537

ACS Infect Dis. 2025 Oct 29. doi: 10.1021/acsinfecdis.5c00570. Online ahead of print.

ABSTRACT

Acinetobacter baumannii, a Gram-negative WHO critical priority pathogen, is an opportunistic bacterial pathogen associated with increasing hospital- and community-acquired infections. The emergence of a multidrug-resistant pathogen, especially the carbapenem-resistant A. baumannii (CRAB), has left us with extremely limited treatment options and, consequently, very high morbidity and mortality rates. As per WHO, the transmissibility of A. baumannii is considered between moderate to high. This review provides a unique comprehensive insight into pathogen's global epidemiology, pathogenesis, host-pathogen interaction, tools to study the pathogen, associated diseases, available treatment options, and how the pathogen is becoming resistant to almost all the treatment options available, thus presenting a holistic picture.

PMID:41163397 | DOI:10.1021/acsinfecdis.5c00570

ACS Infect Dis. 2025 Dec 10. doi: 10.1021/acsinfecdis.5c00582. Online ahead of print.

ABSTRACT

Multidrug-resistant pulmonary infections pose significant therapeutic challenges as treatment options continue to dwindle in the face of rising antimicrobial resistance. Similar challenges arise in the management of wound infections such as those resulting from burn and blast injuries, where resistant pathogens severely limit treatment options. These wounds are further complicated by high microbial loads that exacerbate tissue damage, delay healing, and increase the risk of systemic infection. The escalating threat of antimicrobial resistance highlights the urgent need for innovative therapeutic strategies. This study evaluates the therapeutic potential of a novel topical formulation, azithromycin-bicarbonate (AZM-BIC), for addressing drug-resistant infections in both pulmonary and wound settings. Using murine models of infection in bicarbonate-depleted environments, including lung, blast injury, and burn wound models, topical administration of AZM-BIC enabled the localized delivery of therapeutic concentrations of bicarbonate. In the pulmonary model, AZM-BIC significantly reduced the bacterial burden. In vitro and ex vivo studies revealed AZM-BIC's ability to inhibit biofilm formation, a critical factor in managing chronic infections. In wound infection models, AZM-BIC reduced the bacterial burden and enhanced wound healing. These findings establish AZM-BIC as a promising therapeutic approach, offering a targeted, effective solution for pulmonary infection management and wound care amid the growing threat of antimicrobial resistance. Furthermore, given that azithromycin is a well-established antibiotic and bicarbonate is a physiological component that is safe and well-tolerated, AZM-BIC represents a readily translatable strategy for clinical implementation.

PMID:41367276 | DOI:10.1021/acsinfecdis.5c00582

ACS Infect Dis. 2026 Jan 9;12(1):265-275. doi: 10.1021/acsinfecdis.5c00800. Epub 2025 Dec 2.

ABSTRACT

Colorectal cancer, which originates in the epithelial cells of the colon or rectum, is closely associated with dysbiosis of the gut microbiota. Increasing evidence has shown that Fusobacterium nucleatum plays a significant role in colorectal cancer progression by activating inflammatory responses, modulating the tumor microenvironment, and promoting tumor cell proliferation. Antimicrobial peptides targeting Fusobacterium nucleatum have the potential to serve as more effective and less toxic therapeutic agents compared to chemotherapy drugs. In this study, we systematically evaluated the antibacterial activity of Trp-containing peptides, including natural peptides isolated from the skin secretions of the Chinese brown frog (Rana chensinensis) and their derivatives, which exhibit potent antibacterial activity against Fusobacterium nucleatum with minimal cytotoxicity. Mechanistic investigations using membrane permeability assays and membrane potential-sensitive dyes indicated that Trp-containing peptides exert their antimicrobial effects by disrupting the bacterial membrane structure, increasing membrane permeability, and interfering with membrane potential. In a colorectal cancer mouse model infected with Fusobacterium nucleatum, treatment with Trp-containing peptides significantly alleviated tumor-related symptoms, reduced colonic inflammatory cytokine levels, and alleviated colonic tissue damage, as confirmed by histopathological analysis. Importantly, no apparent toxicity or adverse effects were observed during the treatment. These findings indicate that Trp-containing peptides, as lead compounds, not only exhibit potent antibacterial activity but also attenuate Fusobacterium nucleatum associated colorectal cancer progression, providing critical evidence to support the development of innovative therapeutic strategies with combined antimicrobial and antitumor properties.

PMID:41328741 | DOI:10.1021/acsinfecdis.5c00800

Expert Opin Drug Discov. 2025 Oct 29. doi: 10.1080/17460441.2025.2577996. Online ahead of print.

ABSTRACT

INTRODUCTION: Fluorinated amino acids (FAAs) are at a focal point of two key current strategic areas within drug discovery being both important for the design/development of new small molecule drugs as well as having the potential to be exploited in the rapidly expanding area of peptide-based therapeutics. Their exquisite modularity, synthetic versatility and extensive commercial availability coupled with the robust understanding of the roles that fluorine can play, and which improves their potency and physicochemical properties of drug candidates, has enabled FAAs to be widely used in numerous drug discovery programs.

AREAS COVERED: This review provides an overview of the use of fluorinated amino acids in small drug discovery focusing initially on their impact across a diverse range of therapeutic areas before evaluating methods to access them synthetically. Furthermore, in-depth analyses of programs focusing on the design of thrombin, γ-secretase and FXII inhibitors demonstrate how the strategic introduction of a specific fluorinated amino acid within a molecule can significantly favorably modulate the efficacy and/or the physicochemical properties of the lead through enhancing the electronic/steric interactions with the desired biological target.

EXPERT OPINION: While significant advances have been made enabling access to a broad range of fluorinated amino acids and their derivatives, there are several active on-going research areas in this space. These most notably include developing methods for the synthesis of more-constrained fluorinated bicyclic amino acid derivatives potentially as bioisosteric replacements of aromatic moieties to increase the 3D-dimensionality of a compound while retaining both conformational rigidity and defined orientation of the functional vectors.

PMID:41160102 | DOI:10.1080/17460441.2025.2577996

Methods Enzymol. 2025;721:1-17. doi: 10.1016/bs.mie.2025.08.013. Epub 2025 Sep 22.

ABSTRACT

Biaryl coupling reactions are pivotal in the synthesis of complex therapeutic compounds, such as michelline B, vancomycin and arylomycin A2 derivatives. Synthesizing macrocycles, particularly the 2,2'-disubstituted biaryl-bridged peptide in arylomycin derivatives, present significant challenges, including low yields and the requirement for high transition metal loadings. Recent advances in DNA sequencing and enzyme engineering have facilitated the exploration of biocatalytic transformations. By leveraging enzyme engineering and substrate modifications, we report the development of a biocatalytic process using engineered cytochrome P450 enzymes for the oxidative carbon-carbon bond formation, yielding the biphenolic macrocycles present in arylomycin derivatives, at gram scale. This work underscores the transformative potential of P450 enzymes in synthetic organic chemistry, paving the way for novel pharmaceutical advancements.

PMID:41167838 | DOI:10.1016/bs.mie.2025.08.013

J Am Chem Soc. 2025 Nov 19;147(46):42607-42617. doi: 10.1021/jacs.5c13803. Epub 2025 Nov 4.

ABSTRACT

α,α-Disubstituted α-amino acids (dαAAs) are important building blocks for peptidomimetics as they are strong inducers of helicity and protect against proteolytic degradation. However, de novo discovery of dαAA-containing peptides with genetically encoded libraries is limited due to their poor incorporation efficiency. Here, we report the optimized ribosomal incorporation of multiple achiral dαAAs into peptide libraries and their application to high-throughput (>10¹² members) affinity selection against the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ). This dαAA-based screening methodology discovered potent linear and macrocyclic α-helical peptides with low-to-sub nanomolar binding affinities. Hit peptides were proteolytically stable in serum and cell permeable, allowing for in cellulo antagonism of PPARγ. X-ray crystallography revealed that dαAA-containing peptides bound at the α-helical protein-protein interaction (PPI) interface via an α-helical conformation. This work validates the potential of a dαAA-based, α-helical discovery platform, providing access to new chemical and conformational space to de novo identify novel α-helical peptidomimetics.

PMID:41188059 | PMC:PMC12637311 | DOI:10.1021/jacs.5c13803

ACS Infect Dis. 2025 Oct 13. doi: 10.1021/acsinfecdis.5c00765. Online ahead of print.

ABSTRACT

Bacterial glycans are validated antibiotic targets due to their crucial roles in supporting bacterial fitness and survival. The array of exclusively bacterial monosaccharides and their variable expression across bacterial species and serotypes present challenges in studying these structurally diverse molecules. Probes based on bacterial sugars have emerged as useful tools in metabolic labeling studies. Prior to the metabolic processing of probes by bacteria, most metabolic probes must be transported across the bacterial cell envelope. Probe acetylation has been used as one strategy to ease passive diffusion across the lipophilic cell membrane and relies on deacetylation by esterases within cells before subsequent metabolic processing into glycans is possible. However, inefficient probe deacetylation has the potential to yield artifactual labeling rather than physiological glycan labeling. Here, we systematically explored probe acetylation as a design criterion for metabolic labeling experiments in four bacterial species. Plesiomonas shigelloides, Vibrio vulnificus, and Helicobacter pylori exhibited a strong preference for metabolic incorporation of acetylated probes relative to unprotected probes, whereas Bacteroides fragilis incorporated both unprotected and acetylated probes at comparable levels. Curiously, only B. fragilis had sufficient esterase activity to quantitatively deacetylate a peracetylated monosaccharide probe in situ. These findings suggest the importance of validating acetylated probes on a case-by-case basis to ensure physiologically relevant bacterial glycan labeling.

PMID:41082400 | DOI:10.1021/acsinfecdis.5c00765