Karl Ocius

Microbiol Res. 2025 Jul;296:128160. doi: 10.1016/j.micres.2025.128160. Epub 2025 Mar 29.

ABSTRACT

Regulation of the innate immune response may be an effective strategy to enhance Staphylococcus aureus vaccines. Based on our previous findings that the Listeria peptidoglycan skeleton (pBLP) enhances the immune response through an unknown mechanism, we hypothesized that pBLP provides protection by modulating the innate immune response via trained immunity. In vitro, pBLP increased phagocytosis and inflammatory cytokine levels and elevated the anti-inflammatory cytokine TGF-β following secondary stimulation. In an in vivo model, our findings indicate that pBLP, when administered with a vaccine, protects mice from methicillin-resistant S. aureus challenge and also provides protection against S. aureus CMCC26003 in the absence of antigens. Using an ex vivo model, we demonstrated that pBLP increases markers of trained immunity in peritoneal macrophages. Transcriptome analysis of differentially expressed genes and inhibitor experiments revealed that the trained immunity process induced by pBLP depends on mTOR-HIF-1α and hexokinase 2. This study is the first to demonstrate that pBLP can induce trained immunity. Furthermore, we show that the peptidoglycan skeleton induces a distinct trained immunity phenotype compared to β-glucan, enhancing vaccine protection. Our study provides valuable insights for the design of novel vaccines that integrate both specific and innate immune responses.

PMID:40174361 | DOI:10.1016/j.micres.2025.128160

ACS Nano. 2025 Apr 2. doi: 10.1021/acsnano.5c01930. Online ahead of print.

ABSTRACT

The bacterial cell wall, an essential structure for maintaining cell morphology and protecting against environmental hazards, is predominantly composed of peptidoglycan (PG). This intricate macromolecule undergoes dynamic synthesis and remodeling throughout the cell cycle. Despite its importance, monitoring PG dynamics in live cells, particularly with detailed spatial distribution, poses significant challenges. To this end, we present a series of rhodamine-based fluorogenic probes specifically optimized for real-time and super-resolution imaging of PG synthesis. By fine-tuning the self-aggregation of the probes through the incorporation of hydrophobic linkers, we achieved a substantial reduction in background fluorescence and significant fluorogenicity after labeling. These advancements have enabled us to attain wash-free labeling across a diverse array of bacterial species. Our approach facilitates the direct visualization of PG synthesis patterns, enabling the quantification of septal PG (sPG) synthesis rates in living bacterial cells. Furthermore, it allows for simultaneous imaging of cell division machinery in living cells via both two-dimensional (2D) and three-dimensional (3D) STED microscopy. This study provides a powerful toolkit for investigating the architecture and dynamics of the bacterial cell wall, paving new paths for research on PG-related cellular processes.

PMID:40173278 | DOI:10.1021/acsnano.5c01930

Biochemistry (Mosc). 2025 Jan;90(Suppl 1):S193-S213. doi: 10.1134/S0006297924603757.

ABSTRACT

An important aspect of medical microbiology is identification of the causes and mechanisms of reactivation (resuscitation) of dormant non-sporulating bacteria. In particular, dormant Mycobacterium tuberculosis (Mtb) can cause latent tuberculosis (TB), which could be reactivated in the human organism to the active form of the disease. Analysis of experimental data suggested that reactivation of mycobacteria and reversion of the growth processes include several stages. The initial stage is associated with breakdown of the storage substances like trehalose upon the action of trehalase and with peptidoglycan hydrolysis. Demethylation of tetramethyl porphyrins accumulated in hydrophobic sites (membranes) of the dormant cell also occur in this stage. Metabolic reactivation, starting with cAMP synthesis and subsequent activation of metabolic reactions and biosynthetic processes take place at the stage two as has been shown in the omics studies. Mechanisms of cell reactivation by exogenous free fatty acids via activation of adenylate cyclase and cAMP production have been also suggested. Onset of the cell division is a key benchmark of the third and final stage. Hydrolysis of peptidoglycan as a result of enzymatic action of peptidoglycan hydrolases of the Rpf family is an important process in reactivation of the dormant mycobacteria. Two possible mechanisms for participation of Rpf proteins in reactivation of the dormant bacteria are discussed. On the one hand, muropeptides could be formed as products of peptidoglycan hydrolysis, which could interact with appropriate receptors in bacterial cells transducing activating signal via the PknB phosphotransferase. On the other hand, Rpf protein could presumably change structure of the cell wall, making it more permeable to nutrients and substrates. Both hypotheses were examined in this review. Upon reactivation, independent enzymatic reactions resume their functioning from the beginning of reactivation. Such activation of the entire metabolism occurs rather stochastically, which concludes in combining all biochemical processes in one. This review presents current knowledge regarding biochemical mechanisms of the dormant mycobacteria reactivation, which is important for both fundamental and medical microbiology.

PMID:40164159 | DOI:10.1134/S0006297924603757

Angew Chem Int Ed Engl. 2025 Mar 28:e202503049. doi: 10.1002/anie.202503049. Online ahead of print.

ABSTRACT

Developing selective labeling probes for specific bacterial taxa can not only facilitate the study of target bacteria but also deepen our understanding of the microbial diversity at structural and molecular levels. The availability of such probes, however, remains very limited. In this study, by exploiting the variation of amino acids in peptidoglycan stem peptide, we designed a fluorescent L-amino acid probe and found that it could selectively target the family Lachnospiraceae (a major Gram-positive family in murine gut microbiome) in vivo. The following in vitro test using two Roseburia species validated the probe's labeling. We then discovered that the labeling site was the first amino acid (L-alanine in most bacteria), which links the stem peptide with N-acetylmuramic acid, a process catalyzed by a highly conserved enzyme MurC. Enzyme assay of Roseburia MurC demonstrated its ability to conjugate a fluorescent L-amino acid and other non-L-Ala amino acids to UPD-N-acetylmuramic acid. Subsequent X-ray crystallography analysis uncovered a substantially enlarged inner space in this enzyme, which can partially explain its tolerance to these atypical substrates. The resulting unusual peptidoglycan structures lead to significantly reduced activation of the NOD immune receptors, suggesting a new mechanism for the host to accommodate these highly abundant commensals.

PMID:40152026 | DOI:10.1002/anie.202503049

J Am Chem Soc. 2025 Apr 2;147(13):11189-11198. doi: 10.1021/jacs.4c17913. Epub 2025 Mar 24.

ABSTRACT

The mycobacterial cell envelope plays both infectious and protective roles. Understanding its structure is crucial for unlocking the molecular basis underlying these functions. Studying glycans, the primary components of the cell envelope, is challenging due to their limited native functional handles for chemoselective modification. New labeling methods exploit biorthogonal chemistry, using small molecule mimics that intercept cellular metabolism or late-stage glycan biosynthesis. However, these strategies can have practical limitations, including probe delivery and effectiveness. An ideal small molecule probe should be easily deployed and exploit the critical enzyme-substrate relationships of natural substrates. To this end, we developed a "probegenic" strategy to label mycobacteria. Our approach eliminates the need for explicit substrate mimicry, as the relevant functionality is revealed by a target enzyme. Specifically, we synthesized an azide-substituted trans-β-lactone probe (AzLac), which adopts a substrate-like structure upon covalent enzyme labeling. This probe is incorporated by mycolyltransferases into a core mycobacterial cell envelope glycan, including in the pathogen Mycobacterium tuberculosis. Unlike other probes of the cell envelope, AzLac facilitates selective covalent labeling of the inner leaflet of the mycomembrane. Using Corynebacterium glutamicum mycolyltransferase deletion strains, we implicated Cmt2 as the primary mycolyltransferase target. We leveraged the ability to modify the cell envelope by demonstrating that AzLac could be used to attach a DNA barcode to mycobacteria, which would help track infection dynamics. Thus, we expect AzLac will be a valuable means of monitoring and tracking the mycobacterial cell envelope. Moreover, we anticipate masking and revealing recognition motifs in probes can be applied to diverse cellular targets.

PMID:40126103 | PMC:PMC12989089 | DOI:10.1021/jacs.4c17913

Commun Chem. 2025 Mar 26;8(1):91. doi: 10.1038/s42004-025-01490-6.

ABSTRACT

Peptidoglycan is an essential component of the bacterial cell envelope-a mesh-like macromolecule that protects the bacterium from osmotic stress and its internal turgor pressure. The composition and architecture of peptidoglycan is heterogeneous and changes as bacteria grow, divide, and respond to their environment. Though peptidoglycan has long been studied via LC-MS/MS, the analysis of this data remains challenging as peptidoglycan's unusual composition and branching can't be handled by proteomics software. Here we describe user-friendly open-source tools and a web interface for building peptidoglycan databases, performing MS searches, and predicting the MS/MS fragmentation of muropeptides. We then use Rhizobium leguminosarum to describe a step-by-step strategy for the high-resolution analysis of peptidoglycan. The unprecedented detail of R. leguminosarum's peptidoglycan composition (>250 muropeptides) reveals even the subtlest remodelling between growth conditions. These new and easier to use tools enable more systematic analyses of peptidoglycan dynamics.

PMID:40133660 | PMC:PMC11937551 | DOI:10.1038/s42004-025-01490-6

Org Biomol Chem. 2025 Apr 2;23(14):3459-3464. doi: 10.1039/d5ob00261c.

ABSTRACT

Cell-permeable ubiquitin (Ub) probes labeled with a fluorescent or biotin tag have become effective tools for studying the activity of deubiquitinating enzymes (DUBs) in living cells. However, such probes are usually obtained through total chemical synthesis. In this study, we develop a Sortase A (SrtA)-mediated semi-synthetic strategy for preparing cell-permeable Ub-propargylamide (Ub-PA) with a fluorescent or biotin tag. We demonstrated efficient internalization of the fluorescent Ub-PA probe by live-cell fluorescence confocal microscopy. Additionally, the probe enables selective labeling of distinct DUB families through its attached biotin tag, facilitating subsequent proteomic profiling.

PMID:40085425 | DOI:10.1039/d5ob00261c

J Adv Res. 2025 Mar 12:S2090-1232(25)00152-3. doi: 10.1016/j.jare.2025.03.011. Online ahead of print.

ABSTRACT

INTRODUCTION: Concerns about antibiotic resistance have prompted interest in alternative strategies for enhancing disease resistance, particularly in livestock and poultry production.

OBJECTIVES: This study explored the role of trained immunity in enhancing resistance to Salmonella enterica serovar Typhimurium (S. Typhimurium) infection in mice and chickens.

METHODS: We investigated the effects of probiotics and inactivated pathogenic bacterial strains on host immunity in Toll-like receptor 2-deficient mice (TLR2^-/^-) to assess whether these effects were related to bacterial outer membrane components such as peptidoglycan (PNG), lipoarabinomannan (LAM) and lipoteichoic acid (LTA). Bacterial genomes were evaluated for their ability to enhance the host immune system. Macrophage-depletion models were used to identify the key immune cells involved in trained immunity, with a focus on the cGAS-STING pathway.

RESULTS: Probiotics and inactivated pathogenic strains enhanced host immunity and protected against S. Typhimurium infection. As demonstrated in the TLR2-deficient mice, the effects were not dependent on bacterial outer membrane components. Instead, bacterial genomes played a significant role in activating trained immunity. Macrophages were identified as the primary cells that mediated the response with the cGAS-STING pathway playing a crucial role. The results observed using the mouse models led to investigating the potential application of trained immunity in poultry.

CONCLUSION: Trained immunity activated by probiotics and inactivated bacterial pathogens enhanced resistance against S. Typhimurium infection via macrophage activation and involved the cGAS-STING pathway. These findings highlight the potential of trained immunity as an alternative strategy for disease prevention in both livestock and poultry.

PMID:40086629 | DOI:10.1016/j.jare.2025.03.011

Brain Behav Immun. 2025 Mar 7;127:96-102. doi: 10.1016/j.bbi.2025.03.014. Online ahead of print.

ABSTRACT

Emerging evidence suggests that maternal gut microbiota-derived metabolites and components influence fetal brain development and subsequent neurodevelopment. This study investigates the effects of maternal overexposure to muramyl dipeptide (MDP)-a bacterial peptidoglycan (PGN) motif recognized by Nod2 receptors-on offspring neurodevelopment and behavior. Time-mated C57BL/6J female mice received MDP via drinking water from gestational days 16-19. Nod2 activation in amniotic fluid was assessed using a Nod2 cell-based reporter assay, showing a significant increase in males 24 h after MDP exposure. Gene expression analysis revealed upregulation of PGN transporters in fetal brains, with males showing higher levels of Slc15a1/PepT1, Slc15a2/PepT2, and Slc46a2. No changes in inflammatory or microglia-related markers were found. Behavioral assessments during the juvenile period revealed sex-specific effects: prenatally exposed males showed reduced social interaction, while females exhibited reduced novelty-induced locomotion and impaired social recognition. These behavioral changes were linked to altered expression of synaptic (Dlg4, Ppp1r9b, Darpp-32) and microglial (Trem-2, Cx3cr1) genes in the prefrontal cortex. Our findings underscore the sex-specific effects of maternal PGN overexposure on offspring neurodevelopment, highlighting the potential role of the maternal microbiome in the neurobiology of neurodevelopmental disorders, even in the absence of infection or robust inflammation.

PMID:40058669 | DOI:10.1016/j.bbi.2025.03.014

bioRxiv [Preprint]. 2025 Mar 11:2025.02.26.640348. doi: 10.1101/2025.02.26.640348.

ABSTRACT

Most bacteria are encased in a rigid cell wall peptidoglycan (PG) meshwork. Cell growth requires the activities of both PG synthases and PG hydrolases that cleave bonds within the meshwork enabling its expansion. PG hydrolase activity must be carefully regulated to prevent excessive damage to this protective layer leading to catastrophic lysis. Here, I provide evidence for a novel type of regulation mediated by lipid-linked glycopolymer precursors. The Gram-positive bacterium Bacillus subtilis encodes two functionally redundant PG hydrolases, LytE and CwlO, that are required for growth. Here, I demonstrate that loss of LytR-CpsA-Psr (LCP) enzymes, which enzymatically transfer lipid-linked glycopolymers onto PG, leads to a requirement for lytE for growth. Genetic analysis suggests that this requirement is mediated by the accumulation of these membrane-anchored precursors, where they may interfere with PG hydrolase activity. These results are consistent with models in which polymer transfer influences the position or timing of PG hydrolysis.

PMID:40060662 | PMC:PMC11888478 | DOI:10.1101/2025.02.26.640348