Karl Ocius

Microbiol Immunol. 2026 Jan;70(1):47-51. doi: 10.1111/1348-0421.70025. Epub 2025 Nov 26.

ABSTRACT

To develop vaccine adjuvants from bacterial peptidoglycan (PG) the immunostimulatory activity of lysozyme-solubilized PG derived from Levilactobacillus brevis and Lactiplantibacillus plantarum was investigated. Solubilized PG from both bacteria induced IL-8 in THP-1 cells, and periodate oxidation of L. plantarum PG reduced the activity, suggesting that muramyl dipeptide was partially destroyed. Periodate-oxidized L. plantarum PG showed reduced IL-8 inducing activity in NOD2-expressing cells, while it remained in NOD1-expressing cells, suggesting that γ-d-glutamyl-meso-diaminopimelic acid structure was maintained. All PG preparations stimulated RAW264.7 cells to proliferate, suggesting that they could be potent candidates for vaccine adjuvants.

PMID:41305941 | DOI:10.1111/1348-0421.70025

Biomolecules. 2025 Nov 17;15(11):1609. doi: 10.3390/biom15111609.

ABSTRACT

Although traditionally sidelined by live probiotic effects, Lactobacilli-derived Microbe-Associated Molecular Patterns (MAMPs) are emerging as potent modulators of innate and adaptive immune responses, capable of acting independently of bacterial viability. However, the underlying mechanisms remain incompletely understood. These MAMPs, such as peptidoglycan (PGN), lipoteichoic acid (LTA), and exopolysaccharides (EPSs), interact with pattern recognition receptors (PRRs) like Toll-like receptors (TLRs), initiating immune-signaling cascades that regulate cytokine production and inflammation. Lactobacilli-derived MAMPs exhibit dual immunomodulatory effects: they can enhance pro-inflammatory responses, e.g., interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) under inflammatory contexts, while enhancing regulatory pathways via IL-10 and regulatory T-cell (T_regs) induction in anti-inflammatory settings. Importantly, these immunomodulatory properties persist in the absence of bacterial viability, making MAMPs promising candidates for postbiotic therapies. This opens new avenues for MAMP-based strategies to target inflammation, overcoming the risks associated with live bacterial administration. This review examines the therapeutic relevance of non-viable MAMPs, particularly in inflammatory diseases where they have demonstrated benefits in reducing tissue damage, enhancing gut barrier function, and alleviating disease symptoms. Additionally, we discuss regulatory and translational challenges hindering their clinical implementation, highlighting the need for standardized characterization, a clear safety framework, and strain-specific profiling. Given their ability to fine-tune immune responses, MAMPs represent an emerging strategy for innovative treatments aimed at restoring immune balance and reinforcing host-microbe interactions.

PMID:41301527 | PMC:PMC12650587 | DOI:10.3390/biom15111609

Microbiol Immunol. 2025 Nov 26. doi: 10.1111/1348-0421.70025. Online ahead of print.

ABSTRACT

To develop vaccine adjuvants from bacterial peptidoglycan (PG) the immunostimulatory activity of lysozyme-solubilized PG derived from Levilactobacillus brevis and Lactiplantibacillus plantarum was investigated. Solubilized PG from both bacteria induced IL-8 in THP-1 cells, and periodate oxidation of L. plantarum PG reduced the activity, suggesting that muramyl dipeptide was partially destroyed. Periodate-oxidized L. plantarum PG showed reduced IL-8 inducing activity in NOD2-expressing cells, while it remained in NOD1-expressing cells, suggesting that γ-d-glutamyl-meso-diaminopimelic acid structure was maintained. All PG preparations stimulated RAW264.7 cells to proliferate, suggesting that they could be potent candidates for vaccine adjuvants.

PMID:41305941 | DOI:10.1111/1348-0421.70025

bioRxiv [Preprint]. 2025 Oct 23:2025.04.16.649255. doi: 10.1101/2025.04.16.649255.

ABSTRACT

Microbiota metabolism generates diverse bile acids that are associated with health and disease, but the molecular targets and mechanisms of action for these metabolites have not been fully elucidated. Using bile acid photoaffinity probes and chemoproteomics, we found many protein targets of microbiota-derived secondary bile acids in mammalian cells. Of note, we discovered deoxycholic acid (DCA) binds the transmembrane domain of stimulator of interferon genes (STING), promotes its oligomerization and agonist stimulation of type I interferon signaling ex vivo and antitumor immunity in vivo. Moreover, oral administration of DCA-producing microbiota species enhanced STING agonist antitumor immunity in vivo. This reverse chemical microbiology approach revealed an unpredicted mechanism of action for DCA on STING regulation and suggests specific secondary bile acids and their associated microbiota species may impact the efficacy of STING-targeted therapeutics.

PMID:41278886 | PMC:PMC12633437 | DOI:10.1101/2025.04.16.649255

bioRxiv [Preprint]. 2025 Nov 5:2025.11.02.686169. doi: 10.1101/2025.11.02.686169.

ABSTRACT

The role of the mucosal microbiome in viral infections remains unclear. Genital herpes, caused by herpes simplex virus 1 and 2 (HSV-1 and HSV-2), is among the most prevalent sexually transmitted infections worldwide. Despite evidence linking vaginal Lactobacillus to protection against sexually transmitted viruses, the specific microbial components and mechanisms that mediate this defense are not well understood. Here, we show that multiple cell wall components from diverse gram-positive bacteria, including lactobacilli, inhibit HSV-1 and HSV-2 infection in cells and in a mouse model of genital herpes infection. Peptidoglycan (PG) and lipoteichoic acid (LTA), both major components of the gram-positive bacterial cell wall, significantly reduced HSV infectivity in vitro and improved survival and disease outcomes in mice. We further showed that Lactobacillus crispatus surface layer proteins SlpA and SlpB bind HSV-1 and inhibit infection. Antiviral effects of cell wall components were dose-dependent, relied on intact PG structure, and, in the case of PG and LTA, were independent of TLR2-mediated host signaling. Collectively, our findings identify a species-independent antiviral function for gram-positive bacterial cell wall components against HSV and suggest that the composition of the mucosal microbiome may play an underappreciated role in suppressing mucosal herpes infection in humans.

PMID:41278751 | PMC:PMC12637594 | DOI:10.1101/2025.11.02.686169

bioRxiv [Preprint]. 2025 Oct 14:2025.10.14.682376. doi: 10.1101/2025.10.14.682376.

ABSTRACT

The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis . These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan. They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in the model organism Corynebacterium glutamicum ( Cglu ) using PorH as a model MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu and potentially other members of Mycobacteriales order, coordinate outer membrane protein assembly with PG biogenesis and use proteins to connect the mycomembrane and the cell wall.

SIGNIFICANCE: Diderm bacteria in the Mycobacteriales order have a distinctive outer layer called the mycomembrane. Proteins that reside within the mycomembrane play critical roles in virulence and cell viability. However, how proteins are assembled into the mycomembrane has remained an outstanding question in the field. Here, we investigate the biogenesis of mycomembrane proteins in the model organism Corynebacterium glutamicum . We show that these proteins are inserted into the mycomembrane in a manner that correlates with polar growth and are attached to the cell wall. Many features of these mycomembrane proteins are shared between species in the Mycobacteriales, suggesting that our findings may be conserved in other species within this order.

PMID:41278774 | PMC:PMC12632848 | DOI:10.1101/2025.10.14.682376

Nat Microbiol. 2025 Dec;10(12):3215-3228. doi: 10.1038/s41564-025-02179-1. Epub 2025 Nov 18.

ABSTRACT

Intestinal bile acids are a family of host and microbiota metabolites that can directly inhibit toxin B (TcdB), the primary virulence factor of Clostridioides difficile that causes infectious diarrhoea and colitis. However, the mechanism underlying the inhibition is unclear. Here we used cryogenic electron microscopy and determined the structure of TcdB bound to inhibitory bile acids cholic acid (methyl ester) and taurochenodeoxycholic acid at 2.9 Å and 3.3 Å resolution, respectively. These structures revealed that bile acids lock the C-terminal CROP domain of TcdB in a conformation that allosterically masks the two receptor-binding sites and prevents target cell recognition. Guided by the structure, we synthesized gut-restricted bile acid derivatives, designed to evade the bile acid reuptake transporters within the gut. One of the derivatives, sBA-2, was retained within the gut upon oral dosing and protected mice from toxin-induced C. difficile disease pathology. Our study uncovers the structural basis of inhibition of TcdB by bile acids and its analogues, paving the way for the development of orally deliverable therapeutics against C. difficile.

PMID:41254394 | DOI:10.1038/s41564-025-02179-1

bioRxiv [Preprint]. 2025 Oct 11:2025.10.02.680127. doi: 10.1101/2025.10.02.680127.

ABSTRACT

Antimicrobial resistance (AMR) is an existential threat to modern healthcare; one fueled by selection pressure provided by the use of broad-spectrum antibiotics in medicine and agriculture. As these antibiotics rely on a small set of chemical scaffolds and affect an even smaller number of biological targets, emergent AMR genes can spread through microbiomes to simultaneously inactivate multiple classes and generations of drugs. Long-overlooked for their perceived clinical limitations, antibacterial natural products with taxa-specific activities now present an underexplored source of design principles for precision antibiotics that can selectively eliminate individual microbes and limit community-wide incentives for AMR. Here, we present our re-investigation of one such taxa-specific antibacterial natural product, imacidin, a forgotten inhibitor of cell wall biosynthesis. We show that imacidin is the first natural product inhibitor of the peptidoglycan lipid II flippase MurJ, representing a larger, nascent class of taxa-specific cyclic lipopeptides that offer new leads for precision antibiotics.

PMID:41256586 | PMC:PMC12622048 | DOI:10.1101/2025.10.02.680127

Brain Behav Immun. 2025 Nov 11;131:106175. doi: 10.1016/j.bbi.2025.106175. Online ahead of print.

ABSTRACT

Broad-spectrum β-lactam antibiotics, such as ampicillin, disrupt the commensal gut microbiota and reduce its diversity. However, their potential short-term impact on the translocation of bacterial peptidoglycan (PGN) fragments to the brain and subsequent effects on brain function remain unexplored. In this study, we investigate the effects of a clinically relevant dose of ampicillin on PGN translocation into the brain, gene expression, brain functional connectivity, and social behavior in young adult mice. PGN translocation and gene expression were analyzed at 24-, 48-, and 72-h time points, while behavior, functional connectivity, and gut microbiota were analyzed at 72 h post-exposure. We find that ampicillin increases region-specific PGN translocation into the brain, which correlates with variations in the gene expression levels of PGN transporters and receptors in naïve animals. Antibiotic-treated mice exhibit impaired sociability and social recognition at 72 h post-exposure, which correlate with changes in the expression of synaptic (Syp, Ppp1r9b, Dlg4) and immune (Trem-2) genes in both the prefrontal cortex and striatum, along with disrupted brain functional connectivity. Furthermore, antibiotic-treated mice show an increase in the relative abundance of Gram-negative bacteria at 72 h post-exposure. Mice treated with iE-DAP, a unique PGN fragment from Gram-negative bacteria, exhibit key antibiotic-induced behavioral and molecular traits. Similar to antibiotic-treated mice, iE-DAP-exposed mice show impaired social recognition while maintaining normal motor activity, and reduced expression of synaptic-related genes in the prefrontal cortex and striatum. These findings provide novel insights into the neurobiological mechanisms underlying antibiotic-induced behavioral and functional disruptions and highlight the potential risks to brain health associated with repeated antibiotic use.

PMID:41232619 | DOI:10.1016/j.bbi.2025.106175

Angew Chem Int Ed Engl. 2026 Jan 9;65(2):e19647. doi: 10.1002/anie.202519647. Epub 2025 Nov 10.

ABSTRACT

Tuberculosis (TB), caused by infection with the bacterium Mycobacterium tuberculosis (Mtb), remains one of the most prevalent infectious diseases worldwide. Despite decades of dedicated efforts to develop effective prevention strategies, including vaccines, TB continues to cause significant morbidity and mortality globally. Beyond the Bacille Calmette-Guérin (BCG) vaccine, limited progress has been made to develop more effective TB vaccines. A better understanding of the immunomodulatory roles of key Mtb protein virulence factors is therefore needed for the development of more efficacious protein-based vaccine candidates. Herein, we report a highly efficient method for the semi-synthesis of two native mycobacterial lipoproteins and glycolipoproteins, LprA and Mpt83, respectively. Capitalising on the enhanced reactivity of peptide selenoesters as acyl donors, the 21-23 kDa homogeneously lipidated proteins could be generated in a single ligation step. The homogeneous mycobacterial lipoprotein and glycolipoprotein molecules were shown to be agonists of Toll-like receptor 2 (TLR2) and led to the potent induction of pro-inflammatory cytokines and chemokines in cells. Taken together, this work presents a robust semi-synthetic platform for accessing lipoproteins involved in host-pathogen interactions that can help guide future TB vaccine design.

PMID:41208803 | DOI:10.1002/anie.202519647

Chem Sci. 2025 Oct 28;16(48):23342-23350. doi: 10.1039/d5sc06380a. eCollection 2025 Dec 10.

ABSTRACT

Streptavidin enjoys numerous biotechnological applications due to its extraordinarily high affinity for biotin and the ease of biotinylation of targets through chemical or biological means. However, two main drawbacks limit its use in therapeutic and diagnostic applications: high immunogenicity and endogenous biotin interference. We propose that a mirror-image biotin/streptavidin system could solve these problems due to the minimal immunogenicity of mirror-image (d-) proteins and the expected lower binding affinity between non-natural d-streptavidin and natural d-biotin. To comprehensively address this problem, we first synthesized the l- and d-enantiomers of streptavidin using a three-segment native chemical ligation approach. This synthesis was enabled by temporarily solubilizing an aggregation-prone peptide segment with a Glu-based AlHx 'helping hand' linker. We developed a novel high-efficiency folding protocol and characterized the synthetic proteins by circular dichroism, size-exclusion chromatography, and binding to natural d-(+)-biotin and the mirror-image l-(-)-biotin via isothermal titration calorimetry. We found a 200-million-fold difference in affinity between streptavidin and its matched vs. mismatched biotin enantiomers that renders these systems functionally orthogonal. To gain further insight into how (-)-biotin binds recombinant streptavidin, we solved high-resolution X-ray crystal structures for both the matched and mismatched interactions. This work demonstrates the high degree of stereospecificity of the streptavidin/biotin interaction and the potential utility of a mirror-image biotin/streptavidin system for therapeutic and diagnostic applications.

PMID:41210276 | PMC:PMC12593833 | DOI:10.1039/d5sc06380a