Karl Ocius

Microorganisms. 2026 Apr 18;14(4):917. doi: 10.3390/microorganisms14040917.

ABSTRACT

Staphylococcus aureus remains to be one of the leading causes of global mortality. The most common class of antibiotics used to treat S. aureus infections are next-generation β-lactams (NGBs), as they are highly efficacious and have low adverse effects. NGB resistance in S. aureus is classically attributed to penicillin-binding protein-2a (PBP2a), but previous studies from our group have also implicated an altered expression of penicillin-binding protein-4 (PBP4) with NGB resistance. PBP4 is the sole low-molecular-mass (LMM) PBP present in S. aureus; it is also the only known LMM PBP with transpeptidase activity, giving it the unique ability to bring about peptidoglycan cross-linking. In this article, we review some of the recent findings from our group, which reveal that mutations associated with PBP4 lead to altered protein expression and NGB resistance in both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) backgrounds. We discuss the clinical relevance of PBP4-associated mutations, particularly in methicillin-resistant lacking mec (MRLM) isolates, as well as the synergistic effect of altered PBP4 and GdpP functions. Finally, this review summarizes the potential role played by PBP4 in S. aureus virulence. Together, we highlight the increasing relevance of PBP4 as a mediator of NGB resistance and discuss its potential as an important factor during infection diagnosis and therapy.

PMID:42075313 | PMC:PMC13118971 | DOI:10.3390/microorganisms14040917

Commun Biol. 2026 Apr 30. doi: 10.1038/s42003-026-10124-z. Online ahead of print.

ABSTRACT

Peptidoglycan (PG) is the primary structural component of the bacterial cell wall. However, many bacteria can switch into a wall-deficient "L-form" state, whereby they grow without PG synthesis and become completely resistant to cell-wall-targeting antibiotics. Teichoic acids (TAs) are major glycopolymers of Gram-positive bacteria that are attached to either the PG wall (WTA) or the lipid membrane (LTA). We show that L-form growth does not require either WTA or LTA. However, it does require the TagO protein, which initiates the synthesis of WTA. Inhibiting TagO with the antibiotic tunicamycin triggers a metabolic shift resulting in oxidative damage-mediated cell death, and this is highly synergistic with perturbations of the PG synthetic system. UDP-GlcNAc, a key precursor for both PG and TA synthesis, controls a metabolic switch leading either to balanced growth or oxidative damage. Our results demonstrate the pivotal role of UDP-GlcNAc in the mechanism of killing by cell wall inhibition.

PMID:42062422 | DOI:10.1038/s42003-026-10124-z

Commun Biol. 2026 Apr 25. doi: 10.1038/s42003-026-10072-8. Online ahead of print.

ABSTRACT

Gram staining has guided microbiology for over a century by coloring cells purple or pink, a read-out thought to distinguish monoderms (single membrane, thick peptidoglycan) from diderms (inner and outer membranes with thin wall). Here we show this rule fails repeatedly across Bacillaceae lineages historically deemed "Gram-positive". By combining light and transmission-electron microscopy, antibiotic-sensitivity assays and comparative genomics across 57 strains, we identify "Gram-negative-staining monoderms" lacking outer membrane yet retaining thick peptidoglycan walls. These bacteria lack lipopolysaccharide- and β-barrel assembly-genes and remain highly susceptible to vancomycin and lysozyme (agents normally excluded by diderm envelopes), demonstrating functional monoderm status. Surprisingly, teichoic-acid biosynthetic pathways are patchily distributed and do not predict staining behavior. This discovery calls into question the textbook purple-or-pink dichotomy, decoupling stain color from membrane architecture. Clinically, misidentifying pink-staining Bacillaceae (including emerging pathogens such as Bacillus infantis) risks inappropriate therapy, whereas genome-guided diagnostics enable precise antibiotic stewardship.

PMID:42034801 | DOI:10.1038/s42003-026-10072-8

ACS Omega. 2026 Apr 6;11(15):22841-22851. doi: 10.1021/acsomega.5c12068. eCollection 2026 Apr 21.

ABSTRACT

The stereochemical recognition of the α-methyl group at the d-Ala-d-Ala terminus of peptidoglycan by penicillin-binding proteins (PBPs) has been implicated in shaping the evolutionary divergence of dd-peptidases. Here, we investigate how the β-lactam α-substituent identity influences the conformational dynamics of wild-type Pseudomonas aeruginosa PBP3 using molecular dynamics simulations of covalent acyl-enzyme complexes with CEF_acyl (α-hydro) and its α-methyl derivative, MEC_acyl. Our analysis reveals that the α-methyl group in MEC-PBP3_acyl is accommodated within a defined methyl pocket predominantly composed of conserved residues K297, S349, N351, and V333. Hydration network into the buried active site is disrupted in the MEC-PBP3_acyl complex, which consequently results to the loss of a deacylation-competent geometry of water at K297 toward the β-lactam acyl carbon required for hydrolytic deactivation. Time-resolved analyses of the CEF-PBP3_acyl simulation reveals that the contraction of the active site α-loop is associated with the coupling of water bridge networks that connects the α2 helix active site motif ₂₉₄ STVK ₂₉₇ to a distal salt bridge cluster-the "water sink" (R284 _α1b, D288 _loop, R504 _β4, and D525 _β5) which corroborates crystal structure evidence (PDB ID: 6R3X) [BelliniD., J. Mol. Biol.2019, 431, 3501-3519]. Pocket-based analyses show that the expansion of the methyl pocket into the STVK motif coincides with active site solvation. These dynamic observations are observed to be associated with the shifting salt bridge interactions of R504 _β4 on the α-loop, which may rationalize resistance in R504 mutants. The steric bulk of the α-methyl group in MEC_acyl toward the K297 _α2 side chain disables active site plasticity and consequently impairs the loop mobility required for the influx of water into the active site. These findings provide mechanistic molecular insights into how α-substituent chemistry modulates active site hydration dynamics in PBP3 and support the importance of α-methyl recognition in dd-peptidases. This work also establishes a structural framework for future studies of PBPs and β-lactam drug design relevant to antimicrobial resistance.

PMID:42040443 | PMC:PMC13103851 | DOI:10.1021/acsomega.5c12068

J Immunother Precis Oncol. 2026 Apr 14;9(2):36-45. doi: 10.36401/JIPO-25-31. eCollection 2026 Apr.

ABSTRACT

The class II major histocompatibility complex transactivator (CIITA) is a non-DNA-binding master regulator of major histocompatibility complex class II (MHC-II) gene expression, essential for antigen presentation and adaptive immunity. Functioning as a scaffold, CIITA recruits chromatin remodelers and transcription coactivators to form the MHC-II enhanceosome, facilitating transcriptional activation. CIITA expression is tightly regulated through four promoters and is subject to both cytokine-induced and epigenetic control. Dysregulation of CIITA underpins several immune-related disorders. Its deficiency results in bare lymphocyte syndrome, a severe immunodeficiency. Variants in the CIITA gene have been implicated in autoimmune diseases, graft rejection, and immune dysregulation. Chromosomal translocations involving CIITA are among the most common genomic alterations in some B-cell lymphomas. Additionally, pathogens such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and hepatitis B virus (HBV) exploit CIITA suppression to evade immune surveillance. In oncology, epigenetic silencing of CIITA contributes to MHC-II downregulation and tumor immune evasion. Restoration of CIITA expression enhances tumor immunogenicity, T cell infiltration, and responsiveness to immunotherapy. CIITA also modulates the tumor microenvironment, influences prognosis, and has therapeutic relevance in hematologic and solid tumors. Its multifunctional role positions CIITA as a critical immune regulator and a promising therapeutic target in cancer immunotherapy, antiviral strategies, and immune modulation.

PMID:41994077 | PMC:PMC13078950 | DOI:10.36401/JIPO-25-31

bioRxiv [Preprint]. 2026 Apr 13:2026.04.09.717377. doi: 10.64898/2026.04.09.717377.

ABSTRACT

The multilayered cell envelope of Acinetobacter baumannii is an essential structure that maintains cellular integrity and protects the bacterial cell against external stresses and antibiotics. It consists of an inner membrane, a thin peptidoglycan (PG) layer and an asymmetric outer membrane (OM) enriched in lipooligosaccharide (LOS), whose lipid A moiety is the target of colistin, a last-resource antibiotic. Although lipid A is essential in most Gram-negatives, A. baumannii can survive without LOS through envelope remodeling, particularly in strains producing low levels of the bifunctional penicillin-binding protein PBP1A (encoded by mrcA ) or in D mrcA mutants. Here, we identify a functional interplay between the LD-transpeptidase LdtJ, which generates 3-3 cross-links, and PBP1A, which catalyzes 4-3 transpeptidation during PG synthesis. We show that simultaneous inactivation of both enzymes severely affected growth, viability, morphology, and OM homeostasis. PG analyses revealed that the Δ ldtJ Δ mrcA mutants displays reduced overall cross-linkage and shorter glycan chains, producing a weakened sacculus. Co-immunoprecipitation demonstrated that PBP1A associates with LdtJ, supporting their coordinated activity at sites of PG synthesis. Notably, Δ ldtJ Δ mrcA mutants exhibited the highest recovery frequency of colistin-resistant, LOS-deficient variants compared with wild type or single mutants. Together, our findings demonstrate that coupling between 4-3 and 3-3 transpeptidation is critical for envelope stability in A. baumannii and highlight how disrupting this coordination favors the emergence of colistin resistance. This work identifies a conserved PG remodeling vulnerability that directly links PG integrity to the evolution of antibiotic resistance, offering a new conceptual framework for destabilizing the A. baumannii envelope.

PMID:41993556 | PMC:PMC13082084 | DOI:10.64898/2026.04.09.717377