Karl Ocius

Front Microbiol. 2026 Feb 26;17:1761985. doi: 10.3389/fmicb.2026.1761985. eCollection 2026.

ABSTRACT

Probiotics are well recognized for their ability to modulate host immune responses; however, growing evidence indicates that many of their beneficial effects are mediated by structural components rather than by viable microorganisms. Among these components, probiotic-derived peptidoglycan has emerged as a key immunologically active molecule with a critical role in regulating both innate and adaptive immunity. Although substantial experimental data exist regarding its underlying mechanisms, the context-dependent immunomodulatory and anti-inflammatory functions of peptidoglycan have not been comprehensively integrated. In this review, we provide an up-to-date and comprehensive overview of the molecular and cellular mechanisms that govern the immunoregulatory properties of probiotic-derived peptidoglycan. We first discuss the structural diversity and processing of peptidoglycan and their implications for host recognition via pattern-recognition receptors, particularly Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain proteins 1 and 2 (NOD1/2). We then critically evaluate current evidence supporting the therapeutic potential of probiotic-derived peptidoglycan in infectious diseases, inflammatory bowel disease (IBD), autoimmune disorders, allergic inflammation, and cancer. Collectively, these findings suggest that peptidoglycan holds considerable promise for the development of next-generation microbiota-based immunotherapeutic strategies.

PMID:41834851 | PMC:PMC12980888 | DOI:10.3389/fmicb.2026.1761985

bioRxiv [Preprint]. 2026 Mar 5:2026.03.05.709893. doi: 10.64898/2026.03.05.709893.

ABSTRACT

Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics by blocking drug efflux and hold promise as adjuvant therapeutics in the fight against multidrug resistant pathogenic bacteria. A hurdle in the field has been the lack of selectivity of small molecule EPIs which often display off-target toxicity due to non-specific binding. To tackle this specificity challenge, we aimed to maximize an inhibitor's binding surface area to efflux pumps by designing miniprotein EPIs using computational protein design and an E. coli co-expression assay to screen inhibition in cells. We used S. aureus NorA as a model efflux transporter since it confers drug resistance to fluoroquinolones, puromycin, and other cytotoxic compounds. Starting from a focused miniprotein library of only 86 members, we identified inhibitors in the screen that blocked NorA transport under active efflux conditions in vitro . Our most promising inhibitor I-23 was validated by solving a cryo-EM structure of the miniprotein in complex with NorA, which stabilized the transporter in the outward-open conformation. I-23 has a ferredoxin-like fold with one of its β-hairpins inserted into the substrate binding pocket of NorA and other parts of the globular fold occupying the shallow pocket and making extensive intermolecular contacts with NorA. An arginine residue on the tip of the hairpin loop was positioned near an anionic patch required for NorA antibiotic efflux. The identified structural motifs in this work could be employed to explore the molecular properties of peptidoglycan penetration; full realization of the therapeutic potential of the designed miniprotein inhibitors will require determining the principles for facilitating passage of ∼7 to 8 kDa miniproteins across the peptidoglycan bacterial cell wall.

PMID:41847033 | PMC:PMC12991122 | DOI:10.64898/2026.03.05.709893

Nat Commun. 2026 Mar 13. doi: 10.1038/s41467-026-70567-0. Online ahead of print.

ABSTRACT

In the relentless arms race between bacteria and phages, bacteria have evolved a variety of defense strategies to combat phage infection. However, no system has been previously demonstrated to specifically inhibit phage genomic DNA ejection. Here, we present a bacterial antiphage system, termed HXS, which provides broad-spectrum and robust antiphage activity by interfering with phage DNA entry. The HXS system consists of two radical S-adenosylmethionine (rSAM) enzymes (HxsB and HxsC), a small protein (HxsD), and the effector HxsA with a peptidoglycan-binding domain and five His-Xaa-Ser (HXS) repeats. HxsB/HxsC catalyze rSAM enzyme-dependent maturation of HxsA, including N-terminal processing and a site-specific +8 Da modification, thereby producing a periplasmic effector required for HXS defense. Biochemical evidence supports a model in which the matured effector likely engages incoming DNA electrostatically to arrest entry, establishing an rSAM enzyme-modified protein effector in antiphage defense.

PMID:41826297 | DOI:10.1038/s41467-026-70567-0

Cell Host Microbe. 2026 Apr 8;34(4):672-691.e13. doi: 10.1016/j.chom.2026.02.004. Epub 2026 Feb 27.

ABSTRACT

Early antibiotic exposure increases late-onset sepsis (LOS) risk in preterm infants, potentially via gut dysbiosis. Analyzing 4,938 longitudinal fecal samples from preterm infants in China, the US, and the UK, we identified a differential pace of gut microbiota development among preterm infants. Delayed maturation correlated with over one-third of LOS risk associated with early antibiotic exposure. Deficiency of a bacterial DL-endopeptidase represented a hallmark of delayed microbiota development and correlated with elevated LOS risk. Supplementation with DL-endopeptidase-producing Enterococcus faecium or Limosilactobacillus reuteri activated the NOD2 receptor via muramyl dipeptide (MDP), regulated macrophage differentiation and polarization, restrained hyperinflammation via cylindromatosis (CYLD) induction, and protected neonatal mice from LOS. A pilot randomized controlled trial showed that L. reuteri supplementation enhanced fecal NOD2 activation in preterm infants. These findings link microbiota immaturity and reduced DL-endopeptidase activity to antibiotic exposure and LOS risk and highlight a candidate biomarker that warrants further validation for clinical translation.

PMID:41763213 | DOI:10.1016/j.chom.2026.02.004

Exp Eye Res. 2026 Jun;267:110935. doi: 10.1016/j.exer.2026.110935. Epub 2026 Feb 20.

ABSTRACT

Bacterial keratitis poses a significant threat to ocular health in companion animals, particularly dogs, often leading to corneal ulceration and visual impairment. Muramyl dipeptide (MDP), a conserved bacterial peptidoglycan motif, activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2), yet its contribution to corneal pathogenesis remains incompletely defined. This study demonstrated that MDP induced the NOD2-dependent activation of the RIPK2/NF-κB pathway, triggering pro-inflammatory cytokine production and suppressing the Nrf2-mediated antioxidant response, thereby exacerbating oxidative stress in canine corneal epithelial cells. Furthermore, MDP promoted NLRP3 inflammasome assembly, caspase-1 activation, and gasdermin D-mediated pyroptosis. Inhibition of NOD2 signaling attenuated these effects, highlighting its central role in coordinating inflammatory and oxidative damage. Notably, the natural compound berberine (BBR) effectively suppressed NOD2 activation, restored redox homeostasis, and inhibited pyroptosis in both cellular and canine models of MDP-induced keratitis. These results revealed a previously unrecognized mechanism by which MDP exacerbated corneal damage through NOD2-driven integration of oxidative stress and pyroptosis. We also identified BBR as a multi-target therapeutic agent capable of disrupting this pathogenic cascade. Our findings provided mechanistic insights into the treatment of bacterial keratitis through modulation of innate immune signaling pathways.

PMID:41724227 | DOI:10.1016/j.exer.2026.110935

Mol Microbiol. 2026 Feb 20. doi: 10.1111/mmi.70058. Online ahead of print.

ABSTRACT

Peptidoglycan synthesis and degradation are both essential for bacterial growth, and damaged peptidoglycan must be continuously repaired. In Escherichia coli, peptidoglycan required for cell elongation is synthesized by the Rod complex. Although RodZ is a non-essential component of this complex, its dysfunction leads to aberrant peptidoglycan synthesis, resulting in defects in cell shape and impaired growth. We previously isolated several suppressor mutants that restore growth in cells with impaired RodZ function (RMR cells). Most suppressor mutations mapped to components in the Rod complex other than RodZ. However, one suppressor mutation was identified in sanA, a gene not previously associated with the Rod complex. This mutation, sanA^M27R, represents a loss-of-function allele. Here, we show that SanA is associated with PBP1B, a non-essential yet physiologically important peptidoglycan synthase. Loss of SanA function partially restored the growth of RMR cells, accompanied by enhanced peptidoglycan synthesis and alleviation of structural defects in the cell wall. These findings indicate that SanA contributes to the regulation of peptidoglycan synthesis and cell wall integrity, potentially through functional interplay with PBP1B-dependent pathways.

PMID:41721466 | DOI:10.1111/mmi.70058

Exp Eye Res. 2026 Feb 20:110935. doi: 10.1016/j.exer.2026.110935. Online ahead of print.

ABSTRACT

Bacterial keratitis poses a significant threat to ocular health in companion animals, particularly dogs, often leading to corneal ulceration and visual impairment. Muramyl dipeptide (MDP), a conserved bacterial peptidoglycan motif, activates nucleotide-binding oligomerization domain-containing protein 2 (NOD2), yet its contribution to corneal pathogenesis remains incompletely defined. This study demonstrated that MDP induced the NOD2-dependent activation of the RIPK2/NF-κB pathway, triggering pro-inflammatory cytokine production and suppressing the Nrf2-mediated antioxidant response, thereby exacerbating oxidative stress in canine corneal epithelial cells. Furthermore, MDP promoted NLRP3 inflammasome assembly, caspase-1 activation, and gasdermin D-mediated pyroptosis. Inhibition of NOD2 signaling attenuated these effects, highlighting its central role in coordinating inflammatory and oxidative damage. Notably, the natural compound berberine (BBR) effectively suppressed NOD2 activation, restored redox homeostasis, and inhibited pyroptosis in both cellular and canine models of MDP-induced keratitis. These results revealed a previously unrecognized mechanism by which MDP exacerbated corneal damage through NOD2-driven integration of oxidative stress and pyroptosis. We also identified BBR as a multi-target therapeutic agent capable of disrupting this pathogenic cascade. Our findings provided mechanistic insights into the treatment of bacterial keratitis through modulation of innate immune signaling pathways.

PMID:41724227 | DOI:10.1016/j.exer.2026.110935

Nat Commun. 2026 Feb 24;17(1):1653. doi: 10.1038/s41467-026-68968-2.

ABSTRACT

An unhealthy diet disrupts feeding behavior and the gut microbiota, but whether early-life dietary effects persist, or can be restored later in life, remains unclear. We investigated whether microbiota-targeted interventions (FOS + GOS or Bifidobacterium longum APC1472) could restore early-life high-fat/high-sugar (HFHS) diet-induced feeding alterations in adult female and male mice. HFHS exposure exclusively in early-life induced persistent, sex-specific feeding alterations in adult mice, despite normalized body weight. Early-life HFHS diet reduced hypothalamic cells expressing feeding-related markers (POMC, GHSR, PNOC, NOD2) in adult mice. Females were more vulnerable, with reduced LEPR⁺ cells and disrupted arginine/tryptophan metabolism, while males showed impaired peptidoglycan sensing and steroid metabolism. We show that microbiota interventions restore these effects via distinct mechanisms. FOS + GOS induced extensive microbiome compositional shifts and sex-specific restoration of gut-brain pathways, while B. longum APC1472 induced greater behavioral restoration with minimal microbiome compositional changes. These findings highlight sex-specific vulnerabilities and mechanism-dependent therapeutic potential of microbiota-based interventions after exposure to early-life unhealthy diets.

PMID:41735265 | PMC:PMC12932662 | DOI:10.1038/s41467-026-68968-2

Brain Behav Immun Health. 2026 Feb 5;52:101192. doi: 10.1016/j.bbih.2026.101192. eCollection 2026 Mar.

ABSTRACT

The gut-brain axis is increasingly recognized as contributor to the pathophysiology of brain disorders, in part through its influence on inflammation. Impaired gut health can lead to so-called leaky gut, allowing bacterial cell wall fragments such as peptidoglycan-derived muramyl dipeptide (MDP) to translocate to the circulation. MDP can activate microglia, key mediators of neuroinflammation, via the intracellular receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Although neuroinflammation is a hallmark of "mild" traumatic brain injury (mTBI), the link between leaky gut and mTBI remains largely unexplored. This preliminary prospective study investigated whether mTBI leads to increased MDP levels and NOD2 activation in the acute phase (N = 246; median 106 min post-injury) and at a ∼1-month follow-up visit (N = 140; median 32 days) in an emergency department cohort, relative to healthy controls (HC; N = 31, with N = 26 at ∼1-month). Serum MDP concentration was measured using an indirect competitive ELISA. Engagement of the pro-inflammatory nuclear factor (NF)-κB pathway was measured in NOD2-transfected cells. Additionally, blood interleukin (IL)-6 and IL-10 levels were quantified. Clinical outcome was measured at six months post-injury using the extended Glasgow Outcome Scale and a symptom questionnaire. Linear mixed effects models showed that concentrations of MDP remained stable across visits in both mTBI and HC (P = 0.62), with no significant main effect of group (P = 0.16) or group × visit interaction (P = 0.25). In contrast, for engagement of NF-κB signaling in NOD2-expressing cells, a significant group × visit interaction (P = 0.004) was observed, with an elevation in mTBI relative to HC at ∼1-month post-injury (P = 0.01, Cohen's d = 0.48), but not in the acute phase (P = 0.22, d = 0.22). This elevation was associated with higher IL-6 (β = 0.16, P = 0.02) and IL-10 (β = 0.17, P = 0.006) levels in the acute phase. No associations with clinical outcome were observed. In conclusion, our preliminary null findings for serum MDP do not directly support the emergence of leaky gut in either the acute phase or at ∼1-month following mTBI. However, transient increases in MDP occurring during the first month cannot be ruled out based on our findings. Increased engagement of the NF-κB pathway in NOD2-expressing cells likely reflects (damage-associated) mechanisms other than MDP. Assessment of NF-κB signaling may serve as a useful marker for studying chronic (neuro)inflammation following mTBI, complementing interleukin responses in the acute phase, an avenue warranting further investigation.

PMID:41704660 | PMC:PMC12908069 | DOI:10.1016/j.bbih.2026.101192