Karl Ocius

PLoS Comput Biol. 2026 May 29;22(5):e1014311. doi: 10.1371/journal.pcbi.1014311. Online ahead of print.

ABSTRACT

NOD1 and NOD2, founding members of the NOD-like receptor (NLR) family, play a crucial role in host defense against bacterial infections. Recognition of peptidoglycan-derived ligands triggers ATP-dependent oligomerization of the NACHT domain, exposing the CARD domains that recruit the adaptor protein RIP2 via CARD-CARD interactions to activate the NF-κB signaling cascade. Although NOD1/2-RIP2 interactions and RIP2CARD filament assembly are established, the precise interfaces that stabilize hetero-CARD filaments remain poorly defined. Here, we integrate in silico structural modeling with molecular dynamics (MD) simulations to elucidate structurally compatible arrangements of NOD1-RIP2 and NOD2-RIP2 hetero-CARD filaments. Our results reveal that NOD1CARD subunits form a structurally compatible homomeric scaffold via canonical (type-I-III) interfaces, accommodating multiple tiers of RIP2CARD rings at both filament termini. Meanwhile, the NOD2 tandem CARDs adopt multiple discrete conformations, reflecting a more intricate structural mechanism. In stable filament conformations, tandem CARDs converge at the type-II interface, with RIP2CARD rings stacking onto CARDa (top-down) and CARDb (bottom-up) interfaces, highlighting the structural role of NOD2CARDb in RIP2-mediated CARD-CARD interaction. In silico mutagenesis, involving charge-reversal and alanine scanning at key interfacial residues, disrupts NOD1-RIP2 and NOD2-RIP2 interactions at both top-down and bottom-up interfaces, leading to rapid interface destabilization within 0.1-0.4 μs of simulation. Together, these results reveal conserved and receptor-specific structural mechanisms governing NOD1/2-RIP2 CARD-CARD interactions and provide deeper structural and dynamic insights into the complex structural mechanisms for NLR-mediated inflammatory signaling.

PMID:42213708 | DOI:10.1371/journal.pcbi.1014311

iScience. 2026 May 15;29(6):115975. doi: 10.1016/j.isci.2026.115975. eCollection 2026 Jun 19.

ABSTRACT

The increasing number of immunotherapies developed in the last two decades presents the need for an appropriate animal model to evaluate the efficacy of these treatments. The spontaneous nature of cancer in dogs and the common features they share with human malignancies make the dog a favorable translational model. The major histocompatibility complex (MHC) molecules in dogs are referred to as dog leukocyte antigens (DLA). Here, we introduce two antibodies for the characterization of the DLA class I immunopeptidome from primary canine tumors. We show that up to 55% of the peptides presented by tumor DLA are identical to peptides reported from common HLA class I molecules, displaying striking similarity in length and anchoring positions. Intriguingly, hundreds of these tumor DLA peptides are derived from well-established cancer-associated antigens. In summary, we demonstrate that canine and human MHC class I molecules are highly homologous in their antigen presentation function and peptide repertoire. These findings exhibit promising implications for advancing cancer immunotherapies and their translation from dogs to humans.

PMID:42199926 | PMC:PMC13200046 | DOI:10.1016/j.isci.2026.115975

ACS Infect Dis. 2026 May 22. doi: 10.1021/acsinfecdis.6c00053. Online ahead of print.

ABSTRACT

Bacterial cells are surrounded by a dynamic cell wall which is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass de novo PG biosynthesis, relying instead on PG recycling to sustain growth and division. Legionella pneumophila (hereafter Legionella), the causative agent of Legionnaires' disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in Legionella. Here, two PG recycling gene homologues in the Legionella genome lpg0296 (amgK) and lpg0295 (murU) were identified; chemical biology strategies were used to illuminate incorporation of "click"-PG-probes into whole PG. Copper-free click chemistry with ultrafast tetrazine-NAM probes enabled live-cell PG labeling further supported the use of recycling programs in Legionella. Deletion of amgK abolished PG labeling, while genetic complementation restored labeling. The data suggest that under conditions where de novo peptidoglycan synthesis is blocked, amgK plays a critical role in maintaining cell wall integrity, as its deletion led to increased antibiotic susceptibility and impaired survival in host alveolar macrophages. An intracellular survival assay demonstrated that while PG recycling is not essential for internalization, survival of Legionella within MH-S murine alveolar macrophages requires functional amgK. These findings underscore the essential role of AmgK in Legionella's intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establishing a foundation for developing novel Legionella-specific antibiotic strategies.

PMID:42172232 | DOI:10.1021/acsinfecdis.6c00053

Chem Sci. 2026 May 14. doi: 10.1039/d6sc00548a. Online ahead of print.

ABSTRACT

Bacterial peptidoglycan (PG) serves as a unique bacterial signature for imaging and therapeutic targeting, yet existing chemical probes require high concentrations and involve disruptive washing steps. Herein, we report a general strategy for designing high-performance PG probes by incorporating a fluorine-substituted benzenesulfonamide moiety. The structural modification promotes the binding of probes to PBPs and significantly improves their PG-incorporation efficiency. Using this approach, we developed a full-color probe palette spanning the visible spectrum, enabling wash-free imaging of live bacteria at low concentrations with outstanding signal-to-background ratios. Beyond imaging, this strategy further led to the creation of FluoEosinY, a PG-targeted photosensitizer that effectively inactivates antibiotic-resistant bacteria without inducing resistance. In a mouse model of methicillin-resistant Staphylococcus aureus (MRSA)-infected skin wounds, FluoEosinY-mediated antimicrobial photodynamic therapy not only achieved marked reductions in bacterial burden but also accelerated wound healing, with negligible toxicity to host tissues. This versatile design strategy advances both the mechanistic investigation of bacterial cell wall physiology and the development of precision-targeted therapeutics to combat antibiotic-resistant infections.

PMID:42181058 | PMC:PMC13192278 | DOI:10.1039/d6sc00548a

ACS Infect Dis. 2026 May 21. doi: 10.1021/acsinfecdis.6c00042. Online ahead of print.

ABSTRACT

Glycopeptide antibiotics (GPAs) are the drugs of "last resort" for treating multidrug-resistant Gram-positive infections such as methicillin-resistant Staphylococcus aureus and enterococci. Unfortunately, GPAs are vulnerable to resistance and the dissemination of various types of vancomycin-resistant determinants warrants the discovery of GPAs with novel chemical structures and modes of action (MOA). The overlooked GPAs from the type V subgroup adopt a unique MOA by interrupting cell wall remodeling and are promising lead candidates in antibiotic development. Herein, we describe the discovery of fumamycin from Streptomyces fumanus CGMCC 4.1732 through phylogenetic-guided genome mining and heterologous expression. Fumamycin is the first decapeptide scaffold GPA free of the conserved 4-hydroxyphenylglycine (Hpg) and tryptophan residues, and its peptide scaffold is cross-linked through a unique biaryl ether bond between two 3,5-dihydroxyphenylglycine (Dpg) residues at positions 1 and 3 in the decapeptide scaffold, and in particular, an unprecedented dibenzo-p-dioxin ring between tyrosine at position 6 (Tyr6) and Dpg at position 9 (Dpg9). Gene disruption determines the two P450s, FumD and FumE, are responsible for constructing the dibenzo-p-dioxin ring and the Dpg1-O-Dpg3 cross-link, respectively. Fumamycin represents a new-to-nature GPA chemotype beyond the existing type I-V GPA classification system, it inhibits bacterial growth through a similar manner to that of type V GPAs by binding to peptidoglycan and blocking the activity of autolysins, suggesting a conserved MOA distinct from type I-IV GPAs that inhibit cell wall biosynthesis by binding to d-Alanyl-d-Alanine motif in lipid II, and providing new lead compounds for developing antibiotics to mitigate antimicrobial resistance.

PMID:42166697 | DOI:10.1021/acsinfecdis.6c00042

medRxiv [Preprint]. 2026 May 6:2026.05.05.26352475. doi: 10.64898/2026.05.05.26352475.

ABSTRACT

Lyme disease is a growing and prominent human health problem caused by a group of spirochaetal bacteria that belong to the Borrelia genus. Persistent Lyme disease infection produces a multi-system disorder that may result in severe arthritis, carditis, neurological problems, and even death. Preventing severe disease requires immediate treatment, but current approaches to diagnose Lyme disease are indirect, serology-based assays that may fail early in infection. All Lyme disease-causing Borrelia species shed distinct and unique fragments of their peptidoglycan cell wall during growth. We exploited this fundamental biological process to develop an acute, urine-based diagnostic test. Using a cocktail of unique and highly specific monoclonal antibodies, our ELISA-mediated approach accurately reports on the status of an active, acute infection, in a laboratory animal model of Lyme disease, as well as humans. This rapid, simple, and innovative approach detects an active infection in as few as 3 days of transmission and in 88% of human patients yet to seroconvert-more than ∼2 weeks before serology would be positive.

PMID:42145611 | PMC:PMC13174708 | DOI:10.64898/2026.05.05.26352475

bioRxiv [Preprint]. 2026 May 5:2026.05.04.722707. doi: 10.64898/2026.05.04.722707.

ABSTRACT

Gram-negative bacteria rely on an asymmetric outer membrane (OM) for barrier integrity, with phospholipids confined to the inner leaflet and glycolipids such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS) forming the outer leaflet. Although LPS/LOS was long considered essential, recent findings challenge this view, leaving the mechanistic basis and evolutionary flexibility unclear. Here, we identify lipid asymmetry as a structural checkpoint that governs access to LOS-independent survival. Using Acinetobacter baumannii as a model, we show that disrupting retrograde phospholipid transport and surface phospholipid degradation destabilizes OM lipid balance, creating a permissive state that enables emergence of LOS-deficient, colistin-resistant variants. Integrated lipidomic and transcriptomic analyses reveal a staged remodeling program that reinforces lipoprotein scaffolds, rewires peptidoglycan synthesis, and expands trafficking pathways to stabilize a glycolipid-free envelope. Critically, loss of LOS coincides with sharp repression of PBP1A, and maintaining its activity blocks adaptation, demonstrating interdependence between OM and peptidoglycan homeostasis. We propose a three-state model-basal, permissive, adapted-that explains how envelope architecture gates evolutionary trajectories to antibiotic resistance.

SIGNIFICANCE: Colistin is a last-resort antibiotic targeting lipopolysaccharide (LPS) or lipooligosaccharide (LOS) in Gram-negative pathogens, and many emerging antimicrobials aim to inhibit LPS/LOS biosynthesis and transport. Resistance usually arises via lipid A modification, which preserves LPS/LOS while reducing colistin binding. Resistance to colistin can also arise via complete loss of LOS, which occurs in some Acinetobacter baumannii strains but is constrained in others, such as strain ATCC 17978. Here, we demonstrate that LOS essentiality is not fixed but dictated by outer membrane architecture. Disrupting phospholipid homeostasis creates a permissive envelope that allows LOS-deficient, colistin-resistant variants to emerge, while reducing peptidoglycan synthesis further promotes this state. These findings identify lipid asymmetry as a structural checkpoint in resistance evolution and suggest that preserving envelope homeostasis could limit bacterial escape from colistin and guide strategies for next-generation antibiotic development.

PMID:42146684 | PMC:PMC13174378 | DOI:10.64898/2026.05.04.722707

Nat Commun. 2026 May 9. doi: 10.1038/s41467-026-72752-7. Online ahead of print.

ABSTRACT

The bacterial cell cycle relies on coordinated and dynamic interactions between division and peptidoglycan synthesis proteins. However, visualizing these interactions in vivo remains technically challenging. Here, we established fluorescence-lifetime imaging microscopy combined with Förster resonance energy transfer (FLIM-FRET) as a robust, spatially resolved technique to visualize protein interactions in living Staphylococcus aureus. We set up and validated the method using cytosolic and membrane-anchored control proteins, achieving FRET efficiencies of up to 40%. Using FLIM-FRET, we mapped protein interactions of the glycosyltransferase FtsW within the septal peptidoglycan-synthesizing complex. We confirmed its interaction with the cognate transpeptidase PBP1 and the regulatory protein DivIB. Notably, we found that FtsW also self-interacts, suggesting that septal peptidoglycan synthesis is performed by a complex of multimers, able to synthesize more than one glycan strand. Inhibition of peptidoglycan synthesis by directly targeting PBP1 with the beta-lactam antibiotic imipenem, but not by targeting the lipid II flippase, therefore depleting the FtsW-PBP1 substrate from the outer surface of the cell membrane, weakened FtsW-PBP1 interaction. This suggests that alterations in FtsW interactions result primarily from antibiotic-induced conformational changes or from uncoupling FtsW-PBP1 activities, resulting in the presence of uncrosslinked glycans, rather than merely from a loss of peptidoglycan synthesis activity.

PMID:42106333 | DOI:10.1038/s41467-026-72752-7