Karl Ocius

Immunol Res. 2025 Apr 16;73(1):72. doi: 10.1007/s12026-025-09627-4.

ABSTRACT

The major histocompatibility complex (MHC) encompasses a group of genes critical for immune system regulation. In humans, these molecules are referred to as human leukocyte antigens (HLA) due to their initial discovery in human leukocytes. Class I molecules present antigens to CD8 + T cells, while Class II molecules present to CD4 + T cells. Here we report a patient who had a background of parental consanguinity and a family history suggestive of immunodeficiency. He presented with clinical symptoms including fever, septic arthritis, recurrent moniliasis. Preliminary diagnostic tests revealed hypogammaglobulinemia and CD4 lymphopenia. Further immunological assessment indicated extremely low expression levels of HLA molecules: HLA ABC at 5% and HLA DR at 0%. Genetic analysis showed a mutation in the regulatory factor X5 (RFX5) gene, leading to a combined immunodeficiency diagnosis. Consequently, hematopoietic stem cell transplantation (HSCT) was planned. Regulatory factor X5plays a pivotal role in immune function by transactivating genes critical for the expression of MHC Class I and Class II molecules, as well as beta- 2-microglobulin (B2M). MHC Class I transcription is controlled indirectly by RFX5, and the RFX5 gene mutation in the patient likely caused the markedly reduced expression of HLA ABC in addition to HLA DR. Combined HLA-ABC and HLA-DR expression analyses via flow cytometry may serve as a valuable diagnostic tool for identifying RFX5-related immunodeficiency at an early stage, facilitating timely genetic testing and appropriate clinical management.

PMID:40240550 | DOI:10.1007/s12026-025-09627-4

Bioconjug Chem. 2025 Apr 29. doi: 10.1021/acs.bioconjchem.4c00495. Online ahead of print.

ABSTRACT

Bifunctional trans-cyclooctene (bTCO) with a carbamate or carbonate at the allylic position and tetrazine provide a promising bioorthogonal click chemistry pair for the click-to-release approach, successfully employed in various biotechnological applications. Herein, we demonstrate a simple and straightforward method to synthesize C₂TCO, a symmetrical bTCO derivative with two hydroxyl groups at the allylic positions. The efficiently synthesized C₂TCO at first was selectively functionalized with a fluorophore (C₂TCO-FL), and the conjugate was labeled onto monoclonal antibodies (Ab-C₂TCO-FL). The fluorophore of Ab-C₂TCO-FL was easily removed from the antibody through the mild treatment of tetrazine, enabling multicycle fluorescent bioimaging. Next, an antibody-drug conjugate targeting PD-L1 was prepared using the linker based on C₂TCO. The cytotoxic payload was efficiently released from the antibody upon tetrazine treatment, which induced cellular cytotoxicity.

PMID:40302379 | DOI:10.1021/acs.bioconjchem.4c00495

Biomolecules. 2025 Apr 9;15(4):550. doi: 10.3390/biom15040550.

ABSTRACT

Multitarget drug discovery, including host-directed therapy, is particularly promising for tuberculosis (TB) due to the resilience of Mycobacterium tuberculosis (Mtb) as well as the complexity of the host's immune response. In this proof-of-concept study, we used high-content imaging to test a novel panel of dual glycogen synthase kinase 3 beta (GSK-3β) and histone deacetylase (HDAC) 1 and 6 inhibitor candidates for their efficacy in reducing the growth of green fluorescent protein (GFP)-expressing mycobacteria in human primary macrophages. We demonstrate that all ten test compounds, also including the GSK-3β inhibitor SB415286, exhibit an antimycobacterial effect of 20-60% at low micromolar doses and are non-toxic to host cells. Mtb growth showed a positive correlation with the respective 50% inhibitory concentration (IC50) values of GSK-3β, HDAC1, and HDAC6 in each compound, indicating that compounds with a potent IC50 value for HDAC1, in particular, corresponded to higher antimycobacterial activity. Furthermore, the results from multiparametric flow cytometry and a customized multiplex RNA array demonstrated that SB415286 and selected compounds, C02 and C06, could modulate immune polarization and inflammation in Mtb-infected macrophages involving an enhanced expression of CCL2, IL-10 and S100A9, but a decrease in inflammatory mediators including COX-2, TNF-α, and NFκB. These data suggest that GSK-3β inhibition alone can decrease the intracellular growth of mycobacteria and regulate macrophage inflammation, while dual GSK-3β/HDAC inhibitors enhance this efficacy. Accordingly, the tailored design of dual GSK-3β/HDAC inhibitors could represent an innovative approach to host-directed therapy in TB.

PMID:40305296 | PMC:PMC12024928 | DOI:10.3390/biom15040550

Sci Rep. 2025 Apr 29;15(1):15095. doi: 10.1038/s41598-025-98367-4.

ABSTRACT

Natural Killer (NK) cells can recognize and kill Mycobacterium tuberculosis (Mtb)-infected cells in vitro, however their role after natural human exposure has not been well-studied. To identify Mtb-responsive NK cell populations, we analyzed the peripheral blood of healthy household contacts of active Tuberculosis (TB) cases and source community donors in an endemic region of Port-au-Prince, Haiti by flow cytometry. We observed higher CD8α expression on NK cells in putative resistors (Interferon γ release assay negative; IGRA- contacts) with a loss of CD8α surface expression during household-associated exposure and active TB disease. In vitro assays and CITE-seq analysis of CD8α⁺ NK cells demonstrated enhanced maturity, cytotoxic gene expression, and response to cytokine stimulation relative to CD8α^- NK cells. CD8α⁺ NK cells also displayed dynamic surface expression dependent on MHC class I in contrast to conventional CD8⁺ T cells. Together, these results support a specialized role for CD8α⁺ NK cell populations during Mtb infection correlating with disease resistance.

PMID:40301594 | PMC:PMC12041513 | DOI:10.1038/s41598-025-98367-4

Protein Sci. 2025 May;34(5):e70139. doi: 10.1002/pro.70139.

ABSTRACT

Cofactor-independent racemases and epimerases produce D-amino acids from their L-isomers for a variety of biological processes. These enzymes operate via an unusual mechanism that relies on an active site cysteine thiolate (pK_a ~ 8.5) to deprotonate an amino acid α-carbon (pK_a ~ 29) and are of interest not only because of their biocatalytic potential for D-amino acid production, but also because many play key roles in biology and are antibiotic targets. However, obtaining crystal structures of these enzymes, especially in their closed, substrate- or inhibitor-bound conformations, is difficult. In this work, we characterized diaminopimelic acid (DAP) epimerase from the cyanobacterium Anabaena. DAP epimerase has long been of interest as an antibiotic target as it converts L,L-DAP to D,L-DAP for lysine and peptidoglycan biosynthesis. We solved three crystal structures of this enzyme in its closed, inhibitor-bound conformation, up to a resolution of 1.5 Å. Two structures show the enzyme covalently bound through its catalytic cysteine residues to previously reported aziridine-based inhibitors. One structure unexpectedly shows the enzyme bound to a different compound, D,L-α-methylDAP, presumably produced as a synthetic byproduct. Stereoselective synthesis of L,L- and D,L-α-methylDAP followed by inhibition assays shows that these compounds are slow-binding inhibitors of DAP epimerase. α-MethylDAP inhibitors provide a more accessible alternative to aziridine-based inhibitors to obtain crystal structures of DAP epimerase in its closed conformation. Comparisons of bacterial, cyanobacterial, and plant DAP epimerases provided here offer new insights into functional and structural differences between these enzymes.

PMID:40299312 | PMC:PMC12039745 | DOI:10.1002/pro.70139

Proc Natl Acad Sci U S A. 2025 May 6;122(18):e2416652122. doi: 10.1073/pnas.2416652122. Epub 2025 Apr 29.

ABSTRACT

Extracellular vesicles (EVs) produced by bacteria contain many bacterial-derived molecules, which play an important role in host interactions and as mediators of bacterial communication. However, the role of EVs in interspecies interactions and their physiological and ecological significance are not well understood. In this study, we found that Escherichia coli EVs inhibit the growth of group A Streptococcus (GAS; Streptococcus pyogenes) by inducing defective cell division via the following processes. E. coli EVs first attach to the cell surface of GAS. In EV-attached GAS cells, multiple septa and Z-rings form in close proximity, which clearly differs from the typical cell division process. This is due to inhibition of peptidoglycan (PG) remodeling in the process after septum formation, in which the next cell division is initiated without completion of peripheral PG synthesis. Therefore, cell division proceeds while inducing cell elongation and cell separation failure, leading to growth inhibition. Furthermore, EV alters the expression of approximately 10% of all genes encoded on the GAS genome, and the diverse functions of these gene sets, which include replication, division, and metabolism, suggest that EVs have a variety of biological effects on the targeted bacterial cells. Notably, E. coli EVs significantly decreased the expression of genes involved in representative GAS virulence, such as slo, nga, and hasA, and also markedly attenuated the pathogenicity of GAS in mice. Our findings provide insight into the competitive functions of EVs between different bacterial species, expanding current knowledge on EV-mediated interspecies interactions.

PMID:40299696 | DOI:10.1073/pnas.2416652122

Microorganisms. 2025 Apr 3;13(4):814. doi: 10.3390/microorganisms13040814.

ABSTRACT

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline and neuronal loss, affecting millions worldwide. Emerging evidence highlights the oral microbiome-a complex ecosystem of bacteria, fungi, viruses, and protozoa as a significant factor in cognitive health. Dysbiosis of the oral microbiome contributes to systemic inflammation, disrupts the blood-brain barrier, and promotes neuroinflammation, processes increasingly implicated in the pathogenesis of AD. This review examines the mechanisms linking oral microbiome dysbiosis to cognitive decline through the oral-brain and oral-gut-brain axis. These interconnected pathways enable bidirectional communication between the oral cavity, gut, and brain via neural, immune, and endocrine signaling. Oral pathogens, such as Porphyromonas gingivalis, along with virulence factors, including lipopolysaccharides (LPS) and gingipains, contribute to neuroinflammation, while metabolic byproducts, such as short-chain fatty acids (SCFAs) and peptidoglycans, further exacerbate systemic immune activation. Additionally, this review explores the influence of external factors, including diet, pH balance, medication use, smoking, alcohol consumption, and oral hygiene, on oral microbial diversity and stability, highlighting their role in shaping cognitive outcomes. The dynamic interplay between the oral and gut microbiomes reinforces the importance of microbial homeostasis in preserving systemic and neurological health. The interventions, including probiotics, prebiotics, and dietary modifications, offer promising strategies to support cognitive function and reduce the risk of neurodegenerative diseases, such as AD, by maintaining a diverse microbiome. Future longitudinal research is needed to identify the long-term impact of oral microbiome dysbiosis on cognition.

PMID:40284650 | PMC:PMC12029813 | DOI:10.3390/microorganisms13040814

Angew Chem Int Ed Engl. 2025 Jul;64(27):e202422673. doi: 10.1002/anie.202422673. Epub 2025 May 5.

ABSTRACT

CD59 is an immunomodulatory cell surface receptor associated with human disease. Despite its importance in complement regulation and bacterial pathogenesis, CD59 remains a challenging therapeutic target. Research to date has focused on antibody or protein-based strategies. Here we present a new approach to target CD59 using macrocyclic peptides with low nanomolar affinity for CD59. Through X-ray crystallographic studies and structure-activity relationship (SAR) studies, we identify key interactions that are essential for binding and activity. We find that the macrocyclic peptide CP-06 adopts a beta-hairpin structure and binds CD59 through an intermolecular beta-sheet, mimicking protein-protein interactions of biologically relevant CD59 interaction partners. We create dimeric and lipidated macrocyclic peptide conjugates as enhanced cell-active CD59 inhibitors and show that these probes can be used to modulate both complement-mediated killing of human cells and lytic activity of bacterial virulence factors. Together, our data provide a starting point for future development of macrocyclic peptides to target CD59 activity in diverse cellular contexts.

PMID:40272315 | PMC:PMC12207370 | DOI:10.1002/anie.202422673

Sci Transl Med. 2025 Apr 23;17(795):eadr9091. doi: 10.1126/scitranslmed.adr9091. Epub 2025 Apr 23.

ABSTRACT

Lyme disease, caused by Borrelia burgdorferi in the United States, is an escalating human health problem that can cause severe disease if not properly treated. Doxycycline is the primary treatment option for Lyme disease; however, several concerns are associated with high-dose doxycycline treatment. For example, doxycycline is a broad-spectrum antibiotic and kills beneficial bacteria. Doxycycline is also known to produce unwanted off-target effects in eukaryotic cells. Some at-risk populations such as young children cannot be prescribed doxycycline, and in addition to these shortcomings, the treatment appears to fail in 10 to 20% of cases. We reasoned that safe, alternative therapies may currently exist but have not yet been found because of the challenges associated with drug screening approaches. We screened nearly 500 US Food and Drug Administration-approved compounds using an array of physiological, cellular, and molecular techniques. Top-performing candidates were counter screened to identify compounds that did not affect other bacterial phyla. Piperacillin emerged as a compound that eradicated B. burgdorferi at low-nanomolar concentrations by specifically interfering with the unusual, multizonal peptidoglycan synthesis pattern common to the Borrelia clade. Mechanistic in vitro studies identified the cellular target of piperacillin in B. burgdorferi and produced key insights that may explain both the specificity and efficacy of the compound. Further, in vivo studies using an experimental mouse infection model demonstrated that piperacillin treated animals at a 100-fold lower dose than the effective dose of doxycycline without affecting the murine microbiome. Our findings suggest that piperacillin may offer clinicians another therapeutic option for Lyme disease.

PMID:40267215 | DOI:10.1126/scitranslmed.adr9091

Sci Transl Med. 2025 Apr 23;17(795):eadr2955. doi: 10.1126/scitranslmed.adr2955. Epub 2025 Apr 23.

ABSTRACT

Persistent symptoms after an acute infection is an emerging public health concern, but the pathobiology of such conditions is not well understood. One possible scenario involves the persistence of lingering antigen. We have previously reported that patients with postinfectious Lyme arthritis often harbor the peptidoglycan (PG) cell wall of Borrelia burgdorferi, the Lyme disease agent, in the synovial fluid of their inflamed joints after treatment. However, it is not yet known how B. burgdorferi PG persists, in what form, or if it may play a role in other postinfectious complications after Lyme disease. Using a murine model, we developed a real-time in vivo system to track B. burgdorferi PG as a function of cell wall chemistry and validated our findings using both molecular and cellular approaches. Unlike typical bacterial PG, the unique chemical properties of polymeric B. burgdorferi PG drive murine liver accumulation, where the cell wall material persists for weeks. Kupffer cells and hepatocytes phagocytose and retain B. burgdorferi PG and, although liver occupancy coincides with minimal pathology, both organ-specific and secreted protein profiles produced under these conditions bear some similarities to reported proteins enriched in patients with chronic illness after acute infection. Moreover, transcriptomic profiling indicated that B. burgdorferi PG affects energy metabolism in peripheral blood mononuclear cells. Our findings provide mechanistic insights into how a pathogenic molecule can persist after agent clearance, potentially contributing to illness after infection.

PMID:40267217 | DOI:10.1126/scitranslmed.adr2955

Elife. 2025 Apr 23;14:RP105132. doi: 10.7554/eLife.105132.

ABSTRACT

Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.

PMID:40265569 | PMC:PMC12017771 | DOI:10.7554/eLife.105132

Mol Divers. 2025 Apr 22. doi: 10.1007/s11030-025-11193-8. Online ahead of print.

ABSTRACT

Gram-negative bacterial infections remain a critical global health challenge due to their complex membrane structure and limited treatment options. While peptide deformylase (PDF) inhibitors demonstrate potent activity against Gram-positive pathogens, their efficacy against Gram-negative species is constrained by poor outer membrane permeability. To address this, we rationally designed a novel series of ketone-incorporated compounds with enhanced structural rigidity to improve membrane penetration. Our lead compounds (10a, 10f, 12b) exhibited exceptional activity against Acinetobacter baumannii (MIC₅₀ < 2 μg/mL) and clinically isolated strains (MIC₅₀ < 8 μg/mL), with compound 6 showing particularly potent PDF inhibition (IC₅₀ = 70.8 ± 8.0 nM). The lead compound demonstrated no significant cytotoxicity toward human hepatic stellate cells (LX-2) at the tested concentrations. Molecular docking confirmed their mechanism of action through competitive PDF binding. This work establishes a strategic framework for developing next-generation antibiotics against Gram-negative infections by optimizing membrane permeability while maintaining target inhibition.

PMID:40263230 | DOI:10.1007/s11030-025-11193-8

Cell Death Differ. 2025 Apr 16. doi: 10.1038/s41418-025-01516-5. Online ahead of print.

ABSTRACT

Nucleotide-binding oligomerization domain containing 2 (NOD2) detects conserved fragments of bacterial peptidoglycan in the cytosol and induces innate immune responses. Here, we found that the NOD2 signaling pathway was critically regulated by the deubiquitinating enzyme DUBA. DUBA-deficient macrophages were defective in NOD2 signaling and produced significantly lower amounts of cytokines and chemokines in response to muramyl dipeptide (MDP). DUBA potentiated NOD2-mediated signal transduction by maintaining the protein levels of NOD2 and receptor-interacting protein kinase 2 (RIPK2). Mechanistically, DUBA interacted with NOD2 and RIPK2 and removed K48-linked polyubiquitin chains from them through enzymatic activity, thereby inhibiting the proteasomal degradation of NOD2 and RIPK2. Macrophage-specific ablation of DUBA attenuated MDP-induced systematic inflammation and liver injury in mice. In addition, DUBA deficiency in macrophages rendered mice hypersensitive to DSS-induced colitis and eliminated the protective effect of MDP treatment in colitis. Thus, DUBA acts as an important regulator of NOD2-mediated signaling and innate immune responses.

PMID:40240520 | DOI:10.1038/s41418-025-01516-5

Cell Death Dis. 2025 Apr 4;16(1):248. doi: 10.1038/s41419-025-07599-9.

ABSTRACT

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, making the exploration of metastatic mechanisms crucial for therapeutic advancements. In this study, we identified receptor-interacting protein kinase 2 (RIPK2) as an independent risk factor for poor CRC prognosis. Single-cell RNA sequencing and spatial transcriptomics revealed that a tumor cell cluster with high RIPK2 expression exhibited enhanced metastatic potential, closely linked to bacterial invasion. In vitro and in vivo experiments confirmed that RIPK2 specifically promotes tumor cell migration and invasion, rather than proliferation. Proteomic analysis indicated that RIPK2 knockdown leads to increased proteolysis mediated by ubiquitin, particularly affecting the oncoprotein YAP. Additionally, bacterial invasion of epithelial cells was significantly suppressed in RIPK2 knockdown cells, suggesting a connection to the NOD2-RIPK2 pathway, stimulated by bacterial muramyl dipeptide (MDP). We demonstrated that MDP levels are significantly higher in CRC tissues compared to adjacent non-cancerous tissues, correlating with RIPK2 activation. This activation triggers K63-linked ubiquitination of RIPK2, essential for NF-κB and MAPK pathway activation. Mechanistic studies identified the E3 ubiquitin ligase ITCH as a critical mediator, balancing K63-linked ubiquitination of RIPK2 and K48-linked ubiquitination of YAP, leading to YAP degradation and suppressed CRC metastasis. The stability of YAP could also be disrupted by GSK583, a pharmacological inhibitor of RIPK2, effectively suppressing CRC metastasis. Our findings provide deep insights into RIPK2's role in CRC progression and present a promising target for future therapeutic strategies.

PMID:40185717 | PMC:PMC11971272 | DOI:10.1038/s41419-025-07599-9

iScience. 2025 Mar 16;28(4):112227. doi: 10.1016/j.isci.2025.112227. eCollection 2025 Apr 18.

ABSTRACT

The resistance of Clostridioides difficile to the β-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of C. difficile infections. C. difficile has an original PG structure with a predominance of 3→3 cross-links generated by l,d-transpeptidases (LDTs). C. difficile forms spores and we show that the spore cortex PG contains exclusively 3→3 cross-links. PG and spore cortex of C. difficile cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTs. The d,d-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the l,d-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in C. difficile.

PMID:40224013 | PMC:PMC11986978 | DOI:10.1016/j.isci.2025.112227

Spectrochim Acta A Mol Biomol Spectrosc. 2025 Apr 1;338:126155. doi: 10.1016/j.saa.2025.126155. Online ahead of print.

ABSTRACT

Amino acids, the building blocks of life, exhibit significant chirality, whereas L-amino acids predominate in living organisms and play essential roles in protein synthesis and other biochemical processes. Although less abundant, D-amino acids play critical roles in forming bioactive compounds such as peptidoglycans in bacterial cell walls and find applications in the pharmaceutical industry and materials science. This study uses optical detection techniques to develop a chiral fluorescence sensor based on binaphthol (BINOL). We present the design and synthesis of the (R)-5 probe, which exhibits exclusive selectivity for D-alanine (Ala) among the 20 chiral amino acids, with a detection limit of 10.7 nM. The enantioselectivity of the probe was confirmed through fluorescence spectroscopy and visual inspection, demonstrating its potential for detecting and quantifying D-alanine (D-Ala) in biological samples. This work lays the foundation for developing sensitive diagnostic tools that rely on D-amino acid levels.

PMID:40188573 | DOI:10.1016/j.saa.2025.126155