Karl Ocius

bioRxiv [Preprint]. 2025 Jun 19:2025.06.19.660527. doi: 10.1101/2025.06.19.660527.

ABSTRACT

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane and peptidoglycan layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking outer membrane and peptidoglycan homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in peptidoglycan and incorporate D-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in Acinetobacter baumannii and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates peptidoglycan precursor genes (e.g., dadA , alr ), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a Δ ldtJ Δ mla double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the Δ ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between peptidoglycan remodeling and outer membrane lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through peptidoglycan modification but also by influencing broader regulatory and metabolic networks.

IMPORTANCE: Acinetobacter baumannii is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan layer and outer membrane. While LD-transpeptidases (LDTs) are traditionally known for reinforcing peptidoglycan structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of ldtJ results in pronounced growth defects and abnormal cell morphology - phenotypes that are fully suppressed by disrupting the outer membrane lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between peptidoglycan remodeling and outer membrane lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria and suggest that targeting interconnected stress response pathways could offer novel strategies to undermine bacterial resilience.

PMID:40667300 | PMC:PMC12262596 | DOI:10.1101/2025.06.19.660527

Commun Biol. 2025 Jul 18;8(1):1071. doi: 10.1038/s42003-025-08488-9.

ABSTRACT

Gram-negative (diderm) bacteria possess a multilayered envelope comprising an inner membrane, a thin peptidoglycan (PG) layer and an outer membrane. In Escherichia coli and certain other γ-proteobacteria, including Dickeya dadantii, Braun lipoprotein, Lpp, covalently tethers the outer membrane to PG. Here, we show that in D. dadantii an inner membrane protein, OutB, is covalently attached to PG by the same catalytic mechanism as Lpp. Specifically, two L,D-transpeptidases, Ldt03 and Ldt84, catalyze protein attachment with a preference for muropeptide monomers and dimers, respectively. By altering the Lpp length, we show that the extended Lpp+21 enhances OutB attachment to PG, whereas the truncated LppΔ21 reduces it. Furthermore, we show that the PG-anchoring sequence of OutB tolerates substantial amino acid substitutions and allows PG-tethering of a periplasmic reporter protein, suggesting that other periplasmic and/or membrane proteins may also be tethered to PG in proteobacteria.

PMID:40681671 | PMC:PMC12274547 | DOI:10.1038/s42003-025-08488-9

Nat Commun. 2025 Jul 22;16(1):6737. doi: 10.1038/s41467-025-62097-y.

ABSTRACT

Gut microbiota-derived peptidoglycan fragments (PGNs) are potent inducers of Candida albicans hyphal growth, a key virulence trait for C. albicans pathogenesis in hosts. Herein, we identify the C. albicans oligopeptide transporter 4 (Opt4) as the long-sought major transporter responsible for internalizing a diverse range of natural PGNs into fungal cells. However, contrary to the conventional view, we reveal that blocking the cellular uptake of PGNs does not prevent C. albicans hyphal growth. Instead, we discover that extracellular sensing of PGNs by C. albicans cell surface protein Ssy1 is essential for activating the downstream cAMP-PKA pathway in hyphal signaling. Importantly, the ssy1Δ/Δ mutant, which is defective in PGN-induced hyphal growth, remains unresponsive to the β-lactam-induced PGN storm in the mouse gut. It predominantly maintains yeast morphology and shows no sign of systemic dissemination. These findings establish Ssy1 as a potential anti-virulence target for preventing PGN-induced invasive growth of C. albicans in hosts.

PMID:40695824 | DOI:10.1038/s41467-025-62097-y

Nat Commun. 2025 Jul 21;16(1):6723. doi: 10.1038/s41467-025-62051-y.

ABSTRACT

The peptidoglycan (PG) cell wall is the primary protective layer of bacteria, making the process of PG synthesis a key antibiotic target. Class A penicillin-binding proteins (aPBPs) are a family of conserved and ubiquitous PG synthases that fortify and repair the PG matrix. In gram-negative bacteria, these enzymes are regulated by outer-membrane tethered lipoproteins. However, the molecular mechanism by which lipoproteins coordinate the spatial recruitment and enzymatic activation of aPBPs remains unclear. Here we use single-molecule FRET and single-particle tracking in E. coli to show that a prototypical lipoprotein activator LpoB triggers site-specific PG synthesis by PBP1b through conformational rearrangements. Once synthesis is initiated, LpoB affinity for PBP1b dramatically decreases and it dissociates from the synthesizing enzyme. Our results suggest that transient allosteric coupling between PBP1b and LpoB directs PG synthesis to areas of low peptidoglycan density, while simultaneously facilitating efficient lipoprotein redistribution to other sites in need of fortification.

PMID:40691462 | PMC:PMC12279963 | DOI:10.1038/s41467-025-62051-y

ACS Pharmacol Transl Sci. 2025 Jul 1;8(7):2093-2105. doi: 10.1021/acsptsci.5c00192. eCollection 2025 Jul 11.

ABSTRACT

In a previous study, IOR-160 was identified as a potent dual inhibitor of CK2 and HDAC enzymes. In this study, we evaluated its selectivity and therapeutic potential. IOR-160 exhibited high selectivity for CK2 within a panel of 21 kinases and more widespread inhibitory activity against histone deacetylases (HDAC 1, 2, 3, and 6, low activity for HDAC8). Using a mouse model of triple-negative breast cancer (MDA-MB-231), we further explored its effects on disease progression. Notably, animals treated with IOR-160 exhibited no detectable signs of toxicity or behavioral side effects relative to untreated mice. In a xenograft study, IOR-160 significantly reduced tumor growth (p = 0.0336) and decreased tumor burden (p = 0.0454) compared to the vehicle (DMSO)-treated group. In addition, IOR-160 modulated critical cellular signaling pathways, demonstrated by the inhibition of AKT phosphorylation (p = 0.0175) and a significant increase in acetylated α-tubulin (p = 0.0023), confirming the dual action of IOR-160 in vivo. Furthermore, X-ray crystallography revealed the binding mode of IOR-160 to CK2, showing high conservation compared to that of the known CK2 inhibitor CX-4945. These results suggest that IOR-160 has significant potential as an antitumor agent. Nonclinical and clinical studies become now necessary to validate the efficacy of this new chemical entity as a potential drug.

PMID:40672667 | PMC:PMC12260935 | DOI:10.1021/acsptsci.5c00192

Sci Transl Med. 2025 Jul 16;17(807):eadv0766. doi: 10.1126/scitranslmed.adv0766. Epub 2025 Jul 16.

ABSTRACT

Low tumor mutation burden and an immunosuppressive tumor microenvironment (TME) of colorectal cancers (CRCs) contribute to resistance to immune-checkpoint inhibitors in patients. Understanding the mechanisms of cancer immune evasion will be helpful to develop new therapeutic strategies. Here, leveraging mass spectrometry-based proteomic profiling data and clinical validation, we identified that low sirtuin 2 (SIRT2) expression was associated with improved prognosis and an immune-active TME in CRC. Specifically, genetic knockdown or pharmacological inhibition of SIRT2 resulted in enhanced infiltration and cytotoxicity of CD8⁺ T cells, leading to tumor regression across multiple CRC mouse models and patient-derived organoids. Further in vitro experimental analysis demonstrated that SIRT2 interacted with and deacetylated MutL protein homolog 1 (MLH1) at Lys^402/443/461, thereby preventing MLH1 ubiquitination and degradation. SIRT2 knockdown or inhibition down-regulated MLH1, increasing DNA damage and activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. In addition, both in vivo and in vitro experiments indicated that SIRT2 inhibition stimulated the production of tumor neoantigens and enhanced major histocompatibility complex class I (MHC-I) expression, reprogramming the TME toward an immune-active status and inducing long-lasting immune memory. Last, a combination strategy using SIRT2 inhibitor 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide (AGK2) and anti-programmed cell death protein-1 (PD-1) therapy enhanced immune response, making tumors susceptible to immunotherapy and driving substantial tumor regression in vivo. Our study uncovers a role of SIRT2 in reprogramming TME and underscores the potential of targeting SIRT2 to sensitize CRC to immunotherapy.

PMID:40668890 | DOI:10.1126/scitranslmed.adv0766

bioRxiv [Preprint]. 2025 Jun 10:2025.06.09.658748. doi: 10.1101/2025.06.09.658748.

ABSTRACT

Mammals regulate the localization, composition, and activity of their native microbiota at colonization sites. Lectins residing at these sites influence microbial populations, but their individual functions are often unclear. Intelectins are found in chordates at mucosal barriers, but their functions are not well characterized. We found that mouse intelectin-2 (mItln2) and human intelectin-2 (hItln2) engage and crosslink mucins via carbohydrate recognition. Moreover, both lectins recognize microbes within native microbial communities, including gram-positive and gram-negative isolates from the respiratory and gastrointestinal tracts. This ability to engage mammalian and microbial glycans arises from calcium-coordinated binding of carbohydrate residues within mucus and microbial surfaces. Microbes, but not human cells, bound by mItln2 or hItln2, suffer a loss of viability. These findings underscore the crucial antimicrobial role of mammalian intelectin-2 in mucosal defense, where it plays offensive (microbial killing) and defensive (mucus crosslinking) roles in regulating microbial colonization.

PMID:40661551 | PMC:PMC12259049 | DOI:10.1101/2025.06.09.658748

bioRxiv [Preprint]. 2025 Jun 19:2025.06.19.660527. doi: 10.1101/2025.06.19.660527.

ABSTRACT

The bacterial cell envelope is a critical interface with the environment, particularly in Gram-negative species where the outer membrane and peptidoglycan layers coordinate to maintain structural integrity and resist turgor. Although this coordination is essential for survival, the molecular mechanisms linking outer membrane and peptidoglycan homeostasis remain poorly understood. LD-transpeptidases (LDTs) are enzymes that crosslink peptides in peptidoglycan and incorporate D-amino acids, but their physiological roles are not fully defined. Here, we characterize the activity of the LDT enzyme LdtJ in Acinetobacter baumannii and investigate the consequences of its deletion. Loss of LdtJ disrupts cell morphology, downregulates peptidoglycan precursor genes (e.g., dadA , alr ), and activates the stringent response, including elevated ppGpp levels and dksA upregulation. These defects are fully suppressed in a Δ ldtJ Δ mla double mutant, implicating the outer membrane lipid transport Mla pathway in compensatory regulation. RNA sequencing revealed that transcriptional changes in the Δ ldtJ mutant are reversed in the double mutant, highlighting a functional interplay between peptidoglycan remodeling and outer membrane lipid asymmetry. Our findings suggest that LdtJ contributes to envelope integrity not only through peptidoglycan modification but also by influencing broader regulatory and metabolic networks.

IMPORTANCE: Acinetobacter baumannii is a leading cause of hospital-acquired infections and is highly resistant to antibiotics. Its survival relies on the integrity of the cell envelope, composed of the peptidoglycan layer and outer membrane. While LD-transpeptidases (LDTs) are traditionally known for reinforcing peptidoglycan structure through non-canonical crosslinking, our findings reveal that the LdtJ enzyme also plays a critical role in regulating cellular metabolism and stress responses. Deletion of ldtJ results in pronounced growth defects and abnormal cell morphology - phenotypes that are fully suppressed by disrupting the outer membrane lipid asymmetry transport system, Mla. This genetic interaction uncovers a previously unrecognized link between peptidoglycan remodeling and outer membrane lipid homeostasis. These insights deepen our understanding of envelope coordination in Gram-negative bacteria and suggest that targeting interconnected stress response pathways could offer novel strategies to undermine bacterial resilience.

PMID:40667300 | PMC:PMC12262596 | DOI:10.1101/2025.06.19.660527