Karl Ocius

Front Cell Infect Microbiol. 2025 Feb 11;15:1522114. doi: 10.3389/fcimb.2025.1522114. eCollection 2025.

ABSTRACT

The bacterial cell wall, essential for structural integrity, is synthesized with penicillin-binding proteins (PBPs). Vancomycin-resistant enterococci (VRE) evades vancomycin by replacing D-Ala-D-Ala in their cell wall precursors with D-Ala-D-Lac, reducing the drug's effectiveness. However, how PBPs-which typically use D-Ala-D-Ala as a substrate-adapt to recognize D-Ala-D-Lac remains unclear. Here, we performed Sanger sequencing and alignment of PBP genes from both vancomycin-susceptible and -resistant E. faecalis strains to identify mutations, following amplification by PCR. We then applied homology modeling to assess structural impacts of these changes on PBPs and conducted docking studies to investigate ligand-binding interactions. For the first time, we identified specific adaptations in certain VRE PBPs that may facilitate the D-Ala-D-Lac utilization. We found that PBP1B, PBP2A, PBP3 showed changes, while PBP1A, PBP2B and PBP4 remained unchanged. Notably, a threonine-to-asparagine substitution at location 491 in PBP1B leads to a shift in substrate preference from D-Ala-D-Ala to D-Ala-D-Lac. Similar structural changes in PBP3 suggest that the presence of changed and unchanged PBPs within the same classes suggests compensatory interactions, indicating a teamwork among multiple PBPs. These insights into PBPs provide a deeper understanding of D-Ala-D-Lac utilization in VRE, may be used to develop new therapeutic agents to combat vancomycin resistance.

PMID:40007607 | PMC:PMC11850342 | DOI:10.3389/fcimb.2025.1522114

Bioorg Med Chem. 2025 Apr 1;120:118084. doi: 10.1016/j.bmc.2025.118084. Epub 2025 Jan 29.

ABSTRACT

Antibody-drug conjugates (ADCs) with camptothecin derivatives as payloads have been a hot topic of interest and research since the launch of DS-8201a. As an important component of ADCs, the adequate stability of the linker during circulation and its rapid release at the target site are crucial for the efficient efficacy of ADCs. Although traditional quaternary ammonium ADCs based on dipeptide linkers were highly stable and could be released by specific enzymes, their poor in vitro anti-tumor activity had limited their further exploration. We applied a methylsulfonylethylamine-modified MAC self-elimination system to a valine-alanine linker and constructed a quaternary ammonium ADC (HER2-11) that combines both stability and cleavability. The optimization of the linker effectively improved the in vitro cellular activity of conventional quaternary ammonium ADCs, but the complex intracellular cleavage mechanism of HER2-11 resulted in a weaker anti-tumor activity compared to HER2-GGFG-DXd, which provides great reference value for the continued research of this type of linker in the future.

PMID:39893760 | DOI:10.1016/j.bmc.2025.118084

mBio. 2025 Feb 24:e0395024. doi: 10.1128/mbio.03950-24. Online ahead of print.

ABSTRACT

The cell envelope of gram-negative bacteria consists of two membranes sandwiching the peptidoglycan (PG) cell wall. The outer membrane (OM) contains integrated beta-barrel proteins and has an outer leaflet composed of lipopolysaccharide (LPS). LPS is transported from the inner membrane where it is made to the OM surface by the Lpt system. In the polarly elongating alpha-proteobacterium Brucella abortus, LPS transport has been localized to the polar growth zone and division site. However, LPS transport has not been tracked in live proteobacteria like Escherichia coli that elongate by dispersed incorporation of envelope material along their cell body. Here, we report an investigation into the binding target of fluorescently labeled wheat germ agglutinin (FL-WGA) on E. coli cells that led to the development of a method for visualizing LPS transport. We show that instead of PG or enterobacterial common antigen for which FL-WGA labeling has been used to detect in the past, this probe recognizes LPS modified with a terminal N-acetylglucosamine formed by the defective O-antigen synthesis pathway of laboratory strains of E. coli. This finding enabled the construction of mutants inducible for LPS modification that were used together with FL-WGA labeling to track LPS transport to the cell surface. We show that new LPS is inserted throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously for PG synthesis and OM protein insertion in E. coli, suggesting that LPS transport to the OM is coordinated with these processes.IMPORTANCEGram-negative bacteria like Escherichia coli are surrounded by a multilayered cell envelope that includes an outer membrane (OM) responsible for their high intrinsic resistance to antibiotics. The outer leaflet of this membrane is composed of a glycolipid called lipopolysaccharide (LPS). Here, we report the development of an imaging method to track the transport of LPS to the E. coli outer membrane. The results indicate that transport occurs throughout the cell cylinder and at the division site, but not at the cell poles. A similar pattern was observed previously when cell wall synthesis and the insertion of proteins into the OM were tracked. Our results therefore suggest that LPS transport to the OM is coordinated with other essential processes that underly gram-negative cell envelope biogenesis.

PMID:39992125 | DOI:10.1128/mbio.03950-24

J Oral Biosci. 2025 Mar;67(1):100630. doi: 10.1016/j.job.2025.100630. Epub 2025 Feb 14.

ABSTRACT

OBJECTIVE: Receptor activator of nuclear factor-κB ligand (RANKL) is intimately involved in regulating bone remodeling during osteoclast differentiation and promotion of osteoclast function. Upon binding to its receptor, RANK, RANKL activates various signaling cascades that induce osteoclast differentiation of osteoclast precursor cells into osteoclasts. In the innate immune system, host pattern recognition receptors, such as Toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), detect pathogen-associated molecular patterns and elicit an immune response. The NLR, nucleotide-binding oligomerization domain 2 (NOD2), is known to bind muramyl dipeptide (MDP) and regulate inflammatory responses via nuclear factor-κB (NF-κB). The objective of this study was to investigate the effect of MDP on RANKL stimulation of osteoclast differentiation to elucidate the mechanism of bone resorption in a bacterial infection-induced inflammation model.

METHODS: The extent of osteoclast formation in MDP-stimulated RAW 264.7 cells was assessed using a tartrate-resistant acid phosphatase activity assay. The protein levels of intracellular signaling molecules were assessed by western blotting.

RESULTS: In RAW 264.7 cells, MDP stimulation did not affect the expression of RANK. MDP enhanced the expression of osteoclast-specific proteins, such as nuclear factor of activated T cells 1 (NFATc1) and cathepsin K, which are osteoclast differentiation markers, in RANKL-stimulated RAW 267.4 cells. Furthermore, JSH23, an NF-κB inhibitor, suppressed the expression of NFATc1 after co-stimulation with MDP and RANKL.

CONCLUSION: MDP promoted osteoclast differentiation in RAW 267.4 cells by upregulating the activators, NF-κB and NFATc1, which are important for osteoclast differentiation, through enhancement of the RANKL signaling pathway.

PMID:39956215 | DOI:10.1016/j.job.2025.100630

Chem Commun (Camb). 2025 Mar 6;61(21):4172-4175. doi: 10.1039/d4cc05916f.

ABSTRACT

Here we demonstrate a new approach to fluorogenic labelling, where a cationic hemicyanine (CHyC) exhibits disaggregation-induced emission (DIE) upon undergoing an azide-alkyne "click" reaction. CHyC self-associates and is self-quenched in aqueous buffer over a low micromolar concentration range. When an azido nucleoside (AmdU) or azide-containing cellular DNA is added to CHyC in the presence of Cu(I), a copper-catalysed azide-alkyne cycloaddition drives dye disaggregation, significantly increasing the fluorescence intensity of the probe upon its covalent attachment to modified biomolecules.

PMID:39963867 | DOI:10.1039/d4cc05916f

Elife. 2025 Feb 17;13:RP94982. doi: 10.7554/eLife.94982.

ABSTRACT

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis (Mtb) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR (virR^mut) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb. We employ genetic, transcriptional, proteomics, ultrastructural, and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virR^mut. Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

PMID:39960848 | PMC:PMC11832169 | DOI:10.7554/eLife.94982

Angew Chem Int Ed Engl. 2025 Feb 13:e202420874. doi: 10.1002/anie.202420874. Online ahead of print.

ABSTRACT

Peptidoglycan (PGN) is a complex biopolymer crucial for cell wall integrity and function of all bacterial species. While the strong inflammatory properties of PGN and its derived muropeptides are well-documented in human innate immune responses, adaptive immunity, including antibody responses to PGN, remain inadequately characterized. Microarray technology represents a cost- and time-efficient method for studying such interactions. Our laser-based technology enables the high-throughput synthesis of biomolecules on functionalized glass slides. Here, this on-chip synthesis was developed for PGN fragments, to generate a variety of 216 stem peptides and attach six different glycan moieties that are major structural components of bacterial cell walls. Thereby, 864 PGN fragments from different Gram-negative and Gram-positive species were generated. The arrays were validated with four different monoclonal antibodies against PGN or poly-N-acetyl glucosamine and identified their epitopes. Finally, proof of concept for antibody profiling in patient samples was performed by comparing a panel of well-characterized plasma samples of epidermolysis bullosa (EB) patients suffering from (chronic) wounds with Staphylococcus aureus infection. EB patients show an increased response to the muramyl dipeptide. Therefore, this novel high-throughput PGN glycopeptide microarray technology promises to identify distinct antibody profiles against human microbiomes in diseases, notably in those involving the intestine.

PMID:39945485 | DOI:10.1002/anie.202420874

RSC Adv. 2025 Feb 10;15(6):4526-4531. doi: 10.1039/d4ra07987f. eCollection 2025 Feb 6.

ABSTRACT

Dinitroimidazole (DNIm) was recently identified as a powerful bioconjugation agent that could selectively modify thiol over amine on biomolecules at an ultrahigh speed in an aqueous buffer. However, its derivative containing a DNIm module and a terminal alkyne module failed to construct functional agents bearing a DNIm warhead via the CuAAC reaction. To solve this problem, a heterobifunctional cross-linker was designed and synthesized by linking a DNIm module with an azide module via an oxoaliphatic amido bond spacer arm. Its two modules, DNIm and azide, reacted with a thiol and cyclooctyne, respectively, in an orthogonal way. The cross-linker facilitated the preparation of various functional agents bearing a DNIm warhead via SPAAC reaction and was further applied to protein functionalization (including biotinylation and fluorescence labeling) and oligonucleotide functionalization (including PEGylation, oligonucleotide-peptide and oligonucleotide-protein conjugate). Thus, the cross-linker not only provided convenient access to those functional agents bearing a DNIm warhead but also combined DNIm chemistry with click chemistry of SPAAC to enlarge their respective application range in the bioconjugation field.

PMID:39931413 | PMC:PMC11808663 | DOI:10.1039/d4ra07987f

mSystems. 2025 Feb 12:e0140124. doi: 10.1128/msystems.01401-24. Online ahead of print.

ABSTRACT

The peptidoglycan (PG, or murein) is a mesh-like structure, which is made of glycan polymers connected by short peptides and surrounds the cell membrane of nearly all bacterial species. In contrast, there is no PG counterpart that would be universally found in Archaea but rather various polymers that are specific to some lineages. Methanopyrales and Methanobacteriales are two orders of Euryarchaeota that harbor pseudomurein (PM), a structural analog of the bacterial PG. Owing to the differences between PG and PM biosynthesis, some have argued that the origin of both polymers is not connected. However, recent studies have revealed that the genomes of PM-containing Archaea encode homologs of the bacterial genes involved in PG biosynthesis, even though neither their specific functions nor the relationships within the corresponding inter-domain phylogenies have been investigated so far. In this work, we devised a pangenomic bioinformatic pipeline to identify proteins for PM biosynthesis in Archaea without prior genetic knowledge. The taxonomic distribution and evolutionary relationships of the candidate proteins were studied in detail in Archaea and Bacteria through HMM sequence mining and phylogenetic inference of the Mur domain-containing family, the ATP-grasp superfamily, and the MraY-like family. Our results show that archaeal muramyl ligases are of bacterial origin but diversified through a mixture of horizontal gene transfers and gene duplications. However, in the ATP-grasp and MraY-like families, the archaeal members were not found to originate from Bacteria. Our pangenomic approach further identified five new genes potentially involved in PM synthesis and that would deserve functional characterization.IMPORTANCEMethanobrevibacter smithii is an archaea commonly found in the human gut, but its presence alongside pathogenic bacteria during infections has led some researchers to consider it as an opportunistic pathogen. Fortunately, endoisopeptidases isolated from phages, such as PeiW and PeiP, can cleave the cell walls of M. smithii and other pseudomurein-containing archaea. However, additional research is required to identify effective anti-archaeal agents to combat these opportunistic microorganisms. A better understanding of the pseudomurein cell wall and its biosynthesis is necessary to achieve this goal. Our study sheds light on the origin of cell wall structures in those microorganisms, showing that the archaeal muramyl ligases responsible for its formation have bacterial origins. This discovery challenges the conventional view of the cell-wall architecture in the last archaeal common ancestor and shows that the distinction between "common origin" and "convergent evolution" can be blurred in some cases.

PMID:39936904 | DOI:10.1128/msystems.01401-24

Food Funct. 2025 Jan 29. doi: 10.1039/d4fo06100d. Online ahead of print.

ABSTRACT

This study aimed to investigate the effects of heat-killed Lactiplantibacillus plantarum N1 (HK-N1) and lipoteichoic acid (LTA) derived from it on alleviating insulin resistance by modulating the gut microbiota and amino acid metabolism. High-fat diet (HFD)-fed mice were administered live bacteria or HK-N1, and the results demonstrated that HK-N1 significantly reduced epididymal adipocyte size and serum low density lipoprotein-cholesterol, and improved insulin resistance by increasing the YY peptide and glucagon-like peptide levels. HK-N1 also modulated the gut microbiome composition, enhancing microbiota uniformity and reducing the abundance of Ruminococcus, Oscillospira and norank_f_Mogibacteriaceae. Three main active substances obtained from HK-N1 (membrane protein, peptidoglycan, and lipoteichoic acid) were also used to investigate their potential effects in hyperglycemic zebrafish. Only LTA reduced blood sugar and altered the gut microbiome, particularly reducing Aeromonas, which is positively related to hyperglycemia. Untargeted metabolomics revealed that LTA improved vitamin and amino acid metabolism, thereby alleviating metabolic disorders in zebrafish. Collectively, our findings indicate that HK-N1, primarily through LTA, modulated insulin sensitivity by regulating the gut microbiota and amino acid metabolism, offering a potential therapeutic strategy for insulin resistance and type 2 diabetes mellitus.

PMID:39877991 | DOI:10.1039/d4fo06100d