Karl Ocius

J Clin Invest. 2025 Jan 16;135(2):e179703. doi: 10.1172/JCI179703.

ABSTRACT

Mutations or homozygous deletions of MHC class II (MHC-II) genes are commonly found in B cell lymphomas that develop in immune-privileged sites and have been associated with patient survival. However, the mechanisms regulating MHC-II expression, particularly through genetic and epigenetic factors, are not yet fully understood. In this study, we identified a key signaling pathway involving the histone H2AK119 deubiquitinase BRCA1 associated protein 1 (BAP1), the interferon regulatory factor interferon regulatory factor 1 (IRF1), and the MHC-II transactivator class II transactivator (CIITA), which directly activates MHC-II gene expression. Disruption of the BAP1/IRF1/CIITA axis leads to a functional attenuation of MHC-II expression and MHC-II-dependent immune cell infiltration, leading to accelerated tumor growth in immunocompetent mice. Additionally, we demonstrated that pharmacological inhibition of polycomb repressive complex 1 (PRC1) - which deposits histone H2K119Ub and opposes BAP1 activity - can restore MHC-II gene expression in BAP1-deficient B cell lymphoma cells. These findings suggest that BAP1 may function as a tumor suppressor by regulating the tumor microenvironment and immune response. Our study also establishes the rationale for therapeutic strategies to restore tumor-specific MHC-II expression and enhance immunotherapy outcomes at epigenetic levels in B cell lymphoma treatment.

PMID:39817454 | PMC:PMC11735100 | DOI:10.1172/JCI179703

Acta Pharm Sin B. 2024 Dec;14(12):5341-5356. doi: 10.1016/j.apsb.2024.10.017. Epub 2024 Nov 9.

ABSTRACT

Hydrogen sulfide (H₂S) is a gas signaling molecule with versatile bioactivities; however, its exploitation for disease treatment appears challenging. This study describes the design and characterization of a novel type of H₂S donor-drug conjugate (DDC) based on the thio-ProTide scaffold, an evolution of the ProTide strategy successfully used in drug discovery. The new H₂S DDCs achieved hepatic co-delivery of H₂S and an anti-fibrotic drug candidate named hydronidone, which synergistically attenuated liver injury and resulted in more sufficient intracellular drug exposure. The potent hepatoprotective effects were also attributed to the H₂S-mediated multipronged intervention in lipid peroxidation both at the whole cellular and lysosomal levels. Lysosomal H₂S accumulation and H₂S DDC activation were facilitated by the hydrolysis through the specific lysosomal hydrolase, representing a distinct mechanism for lysosomal targeting independent of the classical basic moieties. These findings provided a novel pattern for the design of optimally therapeutic H₂S DDC and organelle-targeting functional molecules.

PMID:39807337 | PMC:PMC11725097 | DOI:10.1016/j.apsb.2024.10.017

bioRxiv [Preprint]. 2025 Jan 8:2025.01.08.631998. doi: 10.1101/2025.01.08.631998.

ABSTRACT

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PG^Bb) fragments within joint tissues may contribute to the immunopathogenesis, even after appropriate antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PG^Bb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PG^Bb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PG^Bb fragments using a mass spectrometry-based approach. By characterizing the l,d-carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PG^Bb turnover largely occurs at sites of PG^Bb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PG^Bb fragments with 1,6-anhydro bond on their N-acetylmuramyl residues. Stimulation of human cell lines with various synthetic PG^Bb fragments revealed that 1,6-anhydromuramyl-containing PG^Bb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts. We also showed that the activity of the human N-acetylmuramyl-l-alanine amidase PGLYRP2, which reduces the immunogenicity of PG^Bb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PG^Bb fragments and that PG^Bb-induced immunopathology likely results from host sensing of PG^Bb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals.

PMID:39829805 | PMC:PMC11741416 | DOI:10.1101/2025.01.08.631998

Anaerobe. 2025 Jan 13:102939. doi: 10.1016/j.anaerobe.2025.102939. Online ahead of print.

ABSTRACT

The probing of live bacteria via the incorporation of fluorescent D-amino acids (FDAAs) during peptidoglycan synthesis has been shown to be practical for visualizing both gram-positive and gram-negative bacterial species. This study demonstrates the reliability and applications of FDAA labelling for the fluorescent imaging of an obligate anaerobe.

PMID:39814196 | DOI:10.1016/j.anaerobe.2025.102939

Am J Cancer Res. 2024 Dec 15;14(12):5874-5884. doi: 10.62347/NHPV3701. eCollection 2024.

ABSTRACT

Poor oral health is an independent risk factor for upper-aerodigestive tract cancers, including esophageal squamous cell carcinoma (ESCC); thus, good oral health may reduce the risk of ESCC. We previously reported that high expression of Toll-like receptor (TLR) 6, which recognizes peptidoglycan (PGN) from Gram-positive bacteria correlates with a good prognosis after esophagectomy for ESCC. Most beneficial bacteria in the mouth are Gram-positive. We therefore hypothesized that PGN affects cancer cell proliferation and disease progression in ESCC. To test that idea, we assessed the expression of cytokine and chemokine mRNA and protein in eight ESCC cell lines. We also employed a mouse xenograft model to investigate the effect of PGN on ESCC tumor progression in vivo. We then investigated the relationship between the combined expression profiles of TLR6 and C-X-C motif chemokine ligand 10 (CXCL10) in clinical samples and 5-year overall survival (OS) and disease-specific survival (DSS) in ESCC patients after curative esophagectomy. We found that PGN significantly inhibited cell proliferation in six of eight ESCC lines and upregulated CXCL10 production via NF-κB2. In vivo, subcutaneous PGN administration tended to decrease ESCC tumor volume in mice. Combined high expression of TLR6 and CXCL10 correlated with a better prognosis in ESCC patients. This suggests that PGN reduces cell proliferation and tumor progression through a PGN-TLR-CXCL10 cascade, thereby influencing prognosis after esophagectomy for ESCC, and that improving the oral environment could potentially improve the prognosis of ESCC patients after esophagectomy.

PMID:39803645 | PMC:PMC11711533 | DOI:10.62347/NHPV3701

Int J Mol Sci. 2024 Dec 30;26(1):237. doi: 10.3390/ijms26010237.

ABSTRACT

Peptidoglycan (PGN) is a unique component of prokaryotic cell walls with immune-enhancing capacities. Here, we extracted PGN from Corynebacterium glutamicum, a by-product of amino acid fermentation, using the trichloroacetic acid (TCA) method. SDS-PAGE analysis confirmed the presence of PGN, with a band of approximately 28 kDa. Further analysis was conducted through amino acid analysis, FTIR, and MALDI-TOF/TOF MS, and the results showed that the chemical structural monomer of PGN is NAG-(β-1,4-)-NAM-l-Ala-d-Glu-l-Lis-d-Ala. The immune activation effects of PGN were evaluated in a RAW264.7 cell model. Our results showed that PGN could increase the secretion level of NO, ROS, and immune regulatory substances, including TNF-α and IL-1β, and up-regulated the mRNA expression of TNF-α and iNOS. In addition, PGN stimulated the expression of ERK2, MyD88, RIP2, and the related receptor NOD1 in the NF-κB and MAPK pathways. Comparative RNA sequencing was conducted to analyze the gene expression profiles in RAW264.7 cells. KEGG analysis indicated that most of the genes were enriched in the NF-κB, MAPK, and TNF signaling pathways. Taken together, these findings suggest that PGN may have immune-activating potential for the development and application of immune adjuvants. Importantly, the application of PGN also provides a new way to utilize amino acid fermentation by-products.

PMID:39796098 | PMC:PMC11719955 | DOI:10.3390/ijms26010237

Antimicrob Agents Chemother. 2025 Feb 13;69(2):e0117424. doi: 10.1128/aac.01174-24. Epub 2024 Dec 23.

ABSTRACT

Mycobacterium abscessus (Mab) presents significant clinical challenges. This study evaluated the synergistic effects of a β-lactam and β-lactamase inhibitor combination against Mab and explored the underlying mechanisms. Synergy was assessed through MIC tests and time-kill studies, and binding affinities of nine β-lactams and BLIs to eight target receptors (L,D-transpeptidases [LDT] 1-5, D,D-carboxypeptidase, penicillin-binding protein [PBP] B, and PBP-lipo) were assessed using mass spectrometry and kinetic studies. Thermal stability and morphological changes were determined. Imipenem demonstrated high binding affinity to LDTs and PBPs, with extremely low inhibition constants (K_i,app; ≤0.002 mg/L for LDT1-2, ≤0.6 mg/L for PBPs), while cephalosporins, sulopenem, tebipenem, and amoxicillin exhibited moderate to low binding affinity. Durlobactam inactivated Bla_Mab and LDT/PBPs more potently than avibactam. The K_i,apps of durlobactam for PBP B, PBP-lipo, and LDT2 were below clinically achievable unbound concentrations, while avibactam's K_i,app for LDT/PBPs exceeded the clinical concentrations. Single β-lactam treatments resulted in minimal killing (~1 log₁₀ reduction). Although avibactam yielded no effect, combinations with avibactam showed a significant reduction (~4 log₁₀ CFU/mL). Durlobactam alone showed ~2 log₁₀ reduction, and when combined with imipenem or two β-lactams, durlobactam achieved near-eradication of Mab, surpassing the current therapy (amikacin + clarithromycin + imipenem/cefoxitin). Inactivation of PBP-lipo by sulopenem, imipenem, durlobactam, and amoxicillin (with avibactam) led to morphological changes, showing filaments. This study demonstrates the mechanistic basis of combinations therapy, particularly imipenem + durlobactam, in overcoming β-lactam resistance in Mab.

PMID:39714147 | PMC:PMC11823594 | DOI:10.1128/aac.01174-24

Curr Opin Chem Biol. 2024 Dec 18;84:102562. doi: 10.1016/j.cbpa.2024.102562. Online ahead of print.

ABSTRACT

The peptidoglycan manifests as a multifaceted component of the bacterial cell wall. Throughout the lifecycle of the bacterium, the peptidoglycan is deconstructed, rebuilt, and remodeled for bacterial cell growth and replication. Degradation products of the peptidoglycan serve as precursors for cell-wall building blocks via recycling processes and as signaling molecules. Cell-wall recycling and de novo cell-wall synthesis converge biochemically at the cytoplasmic compartment. Peptidoglycan biochemistry is finely tuned to maintain the polymer's functions and is intimately connected to antibiotic-resistance mechanisms. Cell-wall-modifying enzymes present a unique opportunity for the discovery of antibiotics and antibiotic adjuvants. The unique chemical template of the peptidoglycan has been a target of numerous chemical biology approaches for investigating its functions and modulation. In this review, we highlight the current perspective on peptidoglycan research. We present recent efforts to understand the peptidoglycan as a functional component of antibiotic resistance, and as a target for antimicrobial therapy.

PMID:39700530 | DOI:10.1016/j.cbpa.2024.102562

Front Immunol. 2024 Dec 6;15:1451315. doi: 10.3389/fimmu.2024.1451315. eCollection 2024.

ABSTRACT

Lipoteichoic acid (LTA) and peptidoglycan (PGN) are considered as key virulence factors of Staphylococcus aureus, which is a representative sepsis-causing Gram-positive pathogen. However, cooperative effect of S. aureus LTA and PGN on nitric oxide (NO) production is still unclear despite the pivotal roles of NO in initiation and progression of sepsis. We here evaluated the cooperative effects of S. aureus LTA (SaLTA) and muramyl dipeptide (MDP), the minimal structure of PGN, on NO production in both a mouse macrophage-like cell line, RAW 264.7 and mouse bone marrow-derived macrophages (BMMs). Although MDP alone did not affect NO production, MDP potently enhanced SaLTA-induced NO production via the expression of inducible NO synthases. The enhanced NO production was ameliorated in BMMs from TLR2-, CD14-, MyD88-, and NOD2-deficient mice. Moreover, the augmented SaLTA-induced NO production by MDP was attenuated by inhibitors specific for PAFR and MAP kinases. Furthermore, MDP also potently increased SaLTA-induced activities of STAT1, NF-κB, and AP-1 transcription factors, and specific inhibitors for these transcription factors suppressed the elevated NO production. Collectively, these results demonstrated that MDP potentiates SaLTA-induced NO production via TLR2/NOD2/PAFR, MAP kinases signaling axis, resulting in the activation of NF-κB, AP-1 and STAT1 transcription factors.

PMID:39712020 | PMC:PMC11659290 | DOI:10.3389/fimmu.2024.1451315

ACS Catal. 2024 Dec 11;14(24):18786-18798. doi: 10.1021/acscatal.4c05090. eCollection 2024 Dec 20.

ABSTRACT

Remodeling of the pneumococcal cell wall, carried out by peptidoglycan (PG) hydrolases, is imperative for maintaining bacterial cell shape and ensuring survival, particularly during cell division or stress response. The Streptococcus pneumoniae protein Spr1875 plays a role in stress response, both regulated by the VicRK two-component system (analogous to the WalRK TCS found in Firmicutes). Modular Spr1875 presents a putative cell-wall binding module at the N-terminus and a catalytic C-terminal module (Spr1875^MT3) connected by a long linker. Assays of the full-length protein and Spr1875^MT3 with PG-based synthetic substrates by liquid chromatography/mass spectrometry revealed Spr1875 as an l,d-endopeptidase, renamed VldE (for VicRK-regulated l,d-endopeptidase), which hydrolyzed the cross-linked stem peptide in the PG. Remarkably, we observed asymmetric turnover with specific recognition of the acceptor peptide strand. Localization experiments showed that the protein is directed to the septum, which suggests that muralytic activity could be required for pneumococcal growth under stress conditions. Our findings, based on six high-resolution X-ray crystallographic structures and molecular-dynamics simulations, reveal two states for VldE^MT3. The protein transitions between a noncatalytic state that binds up to four zinc ions, thus behaving as a Zn²⁺ reservoir, and a catalytic state that performs the hydrolytic reaction with a single zinc ion. Furthermore, computational studies provide insight into the mechanism of catalytic-water activation and nucleophilic attack on the specific scissile peptide bond of the asymmetric cross-linked PG.

PMID:39722888 | PMC:PMC11667670 | DOI:10.1021/acscatal.4c05090

JCI Insight. 2024 Dec 10:e186346. doi: 10.1172/jci.insight.186346. Online ahead of print.

ABSTRACT

Peptidoglycans (PGNs) are structural polymers of the bacterial cell wall and a common microbial molecular pattern encountered by our immune system daily. Low levels of PGNs are constitutively present in the systemic circulation in humans and elevate during inflammatory pathologies. Since all known PGN sensors are intracellular, PGN internalization is a prerequisite for the initiation of cellular immune responses. Here we report the mechanisms controlling the recognition and uptake of polymeric PGNs by circulating human mononuclear phagocytes. We found that complement C3 and C4 opsonins govern PGN recognition and internalization, but no single opsonin is indispensable due to multiple uptake redundancies. We observed a bimodal internalization of polymeric PGNs with distinct requirements for complement C4. At low PGN concentrations, C3 mediated PGN recognition by surface receptors while the efficient internalization of PGN polymers critically required C4. Supraphysiologic PGN concentrations triggered a secondary uptake modality that was insensitive to C4 and mediated instead by C3 engagement of complement receptors 1 and 3. To our knowledge this is the first description of non-overlapping C3 and C4 opsonophagocytoses working in parallel. Controlling these uptake mechanisms has the potential to modulate PGN clearance and/or the dysregulated immune responses during bacterial infections.

PMID:39656526 | DOI:10.1172/jci.insight.186346

ACS Infect Dis. 2024 Dec 4. doi: 10.1021/acsinfecdis.4c00370. Online ahead of print.

ABSTRACT

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC₅₀ values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, k_inact/K_I, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, k_inact/K_I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell k_inact/K_I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro k_inact/K_I data and show a comparable relationship to previously established IC₅₀ values. These results support the validity of our in vivo k_inact/K_I method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

PMID:39628314 | DOI:10.1021/acsinfecdis.4c00370

J Med Chem. 2024 Dec 5. doi: 10.1021/acs.jmedchem.4c02139. Online ahead of print.

ABSTRACT

Helicobacter pylori (H. pylori) infection is characterized by the complex interplay between H. pylori and gastric disorders. It has been established that NOD1 can be activated by the peptidoglycan (PGN) present in the cell wall of H. pylori, serving as a key mediator of inflammation and initiating the RIP2/NF-κB and MAPK inflammatory signaling pathways. In this article, we reported on the development of a 2-chloroquinazolin-4-ol derivative 66 as a potent and selective antagonist of both human and mouse NOD1, which effectively inhibited the expression of inflammatory cytokines (IL-6, TNF-α) and chemokines (CXCL1, CXCL8) in immune and epithelial cells, as well as inflammatory cytokines (KC, IL-6) in a H. pylori-induced murine model of gastritis following oral administration. This study laid a foundation for treating gastritis induced by H. pylori infection.

PMID:39637404 | DOI:10.1021/acs.jmedchem.4c02139