Karl Ocius

Front Immunol. 2023 Oct 5;14:1242659. doi: 10.3389/fimmu.2023.1242659. eCollection 2023.

ABSTRACT

Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses. This review will describe how NOD1 and NOD2 detect microbes and cellular stress and detail the molecular mechanisms that regulate activation and signaling while highlighting new evidence and the impact on inflammatory disease pathogenesis.

PMID:37869013 | PMC:PMC10585062 | DOI:10.3389/fimmu.2023.1242659

Chem Commun (Camb). 2023 Oct 26;59(86):12859-12862. doi: 10.1039/d3cc02932h.

ABSTRACT

Epoxides are an established class of electrophilic alkylating agents that react with nucleophilic protein residues. We report αβ,α'β'-diepoxyketones (DEKs) as a new type of mechanism-based inhibitors of nucleophilic cysteine enzymes. Studies with the L,D-transpeptidase Ldt_Mt2 from Mycobacterium tuberculosis and the main protease from SARS-CoV-2 (M^pro) reveal that following epoxide ring opening by a nucleophilic cysteine, further reactions can occur, leading to irreversible alkylation.

PMID:37815791 | PMC:PMC10601815 | DOI:10.1039/d3cc02932h

mBio. 2023 Oct 13;14(5):e0137923. doi: 10.1128/mbio.01379-23. Online ahead of print.

ABSTRACT

The peptidoglycan layer is a defining characteristic of bacterial cells, providing them with structural support and osmotic protection. In Escherichia coli, this layer is linked to the outer membrane via the abundant membrane-anchored protein Lpp, known as Braun's lipoprotein, with LD-transpeptidases LdtA, LdtB, and LdtC catalyzing the attachment. However, one distinctive member of the YkuD-type transpeptidase family, LdtF (recently renamed DpaA), carries out the opposite reaction of detaching Lpp from the peptidoglycan layer. In this study, we report the crystal structure of DpaA, which reveals the enzyme's ability to cleave, rather than form, the Lpp-peptidoglycan linkage. Assays with purified peptidoglycan-Lpp as the substrate and chemically synthesized compounds suggest that DpaA's shallow L-shaped active site can only accommodate and cleave the peptidoglycan-Lpp cross-link with a constrained conformation. This study provides insights into how homologous Ldt enzymes can perform opposing chemical reactions. IMPORTANCE Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

PMID:37830798 | PMC:PMC10653827 | DOI:10.1128/mbio.01379-23

Med Microbiol Immunol. 2023 Dec;212(6):421-435. doi: 10.1007/s00430-023-00782-9. Epub 2023 Oct 5.

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for a range of severe infections, such as skin infections, bacteremia, and pneumonia. Due to its antibiotic-resistant nature, current research focuses on targeting its virulence factors. Sortase A (SrtA) is a transpeptidase that anchors surface proteins to the bacterial cell wall and is involved in adhesion and invasion to host cells. Through fluorescence resonance energy transfer (FRET), we identified echinacoside (ECH), a natural polyphenol, as a potential SrtA inhibitor with an IC₅₀ of 38.42 μM in vitro. It was demonstrated that ECH inhibited SrtA-mediated S. aureus fibrinogen binding, surface protein A anchoring, and biofilm formation. The fluorescence quenching assay determined the binding mode of ECH to SrtA and calculated the K_A-binding constant of 3.09 × 10⁵ L/mol, demonstrating the direct interaction between the two molecules. Molecular dynamics simulations revealed that ECH-SrtA interactions occurred primarily at the binding sites of A92G, A104G, V168A, G192A, and R197A. Importantly, the combination of ECH and vancomycin offered protection against murine models of MRSA-induced pneumonia. Therefore, ECH may serve as a potential antivirulence agent against S. aureus infections, either alone or in combination with vancomycin.

PMID:37796314 | DOI:10.1007/s00430-023-00782-9

Methods Mol Biol. 2024;2727:193-204. doi: 10.1007/978-1-0716-3491-2_15.

ABSTRACT

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria.

PMID:37815718 | DOI:10.1007/978-1-0716-3491-2_15

mBio. 2023 Oct 13:e0137923. doi: 10.1128/mbio.01379-23. Online ahead of print.

ABSTRACT

The peptidoglycan layer is a defining characteristic of bacterial cells, providing them with structural support and osmotic protection. In Escherichia coli, this layer is linked to the outer membrane via the abundant membrane-anchored protein Lpp, known as Braun's lipoprotein, with LD-transpeptidases LdtA, LdtB, and LdtC catalyzing the attachment. However, one distinctive member of the YkuD-type transpeptidase family, LdtF (recently renamed DpaA), carries out the opposite reaction of detaching Lpp from the peptidoglycan layer. In this study, we report the crystal structure of DpaA, which reveals the enzyme's ability to cleave, rather than form, the Lpp-peptidoglycan linkage. Assays with purified peptidoglycan-Lpp as the substrate and chemically synthesized compounds suggest that DpaA's shallow L-shaped active site can only accommodate and cleave the peptidoglycan-Lpp cross-link with a constrained conformation. This study provides insights into how homologous Ldt enzymes can perform opposing chemical reactions. IMPORTANCE Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

PMID:37830798 | DOI:10.1128/mbio.01379-23

Chem Commun (Camb). 2023 Oct 26;59(86):12859-12862. doi: 10.1039/d3cc02932h.

ABSTRACT

Epoxides are an established class of electrophilic alkylating agents that react with nucleophilic protein residues. We report αβ,α'β'-diepoxyketones (DEKs) as a new type of mechanism-based inhibitors of nucleophilic cysteine enzymes. Studies with the L,D-transpeptidase Ldt_Mt2 from Mycobacterium tuberculosis and the main protease from SARS-CoV-2 (M^pro) reveal that following epoxide ring opening by a nucleophilic cysteine, further reactions can occur, leading to irreversible alkylation.

PMID:37815791 | PMC:PMC10601815 | DOI:10.1039/d3cc02932h

Eur J Intern Med. 2023 Oct 4:S0953-6205(23)00347-3. doi: 10.1016/j.ejim.2023.10.002. Online ahead of print.

ABSTRACT

The prevalence of overweight, obesity, type 2 diabetes, metabolic syndrome and steatotic liver disease is rapidly increasing worldwide with a huge economic burden in terms of morbidity and mortality. Several genetic and environmental factors are involved in the onset and development of metabolic disorders and related complications. A critical role also exists for the gut microbiota, a complex polymicrobial ecology at the interface of the internal and external environment. The gut microbiota contributes to food digestion and transformation, caloric intake, and immune response of the host, keeping the homeostatic control in health. Mechanisms of disease include enhanced energy extraction from the non-digestible dietary carbohydrates, increased gut permeability and translocation of bacterial metabolites which activate a chronic low-grade systemic inflammation and insulin resistance, as precursors of tangible metabolic disorders involving glucose and lipid homeostasis. The ultimate causative role of gut microbiota in this respect remains to be elucidated, as well as the therapeutic value of manipulating the gut microbiota by diet, pre- and pro- synbiotics, or fecal microbial transplantation.

PMID:37802720 | DOI:10.1016/j.ejim.2023.10.002

Methods Mol Biol. 2024;2727:153-158. doi: 10.1007/978-1-0716-3491-2_13.

ABSTRACT

Sortases are highly conserved enzymes with endopeptidase and transpeptidase activities in Gram-positive bacteria. Sortase A cleaves within an LPXTG-motif and covalently crosslinks cell wall proteins to become anchored to the peptidoglycan of the cell wall. We showed that a peptide cleaved by sortase A from the C-terminus (C-pep) of the LPXTG-adhesin SspA intercalates in the cell membrane. Nested in the membrane, this C-pep docks with the intramembrane sensor histidine kinase, SraS, to activate the response regulator, SraR. SraR signals that the C-pep has been cleaved as an indicator of the fidelity of sortase A processing. SraSR also signals that key LPXTG-proteins in concert with lipoteichoic acid engage the mucin, MUC5B, which elicits a different transcriptional response than the binding of other salivary constituents. To visualize the C-pep intercalating in the cell membrane in vivo, we used Structured Illumination Microscopy (SIM). And to show that the C-pep complexes with SraS, we used bimolecular fluorescence experiments. The C-pep and SraS were each expressed with one or the other half of yellow fluorescence protein (YFP). Reconstitution of the complete YFP signal indicated that the C-pep and SraS interacted at molecular distances within the cell membrane in vivo. Using these imaging protocols, we learned that the C-pep functions as a signaling molecule within the cell membrane of the streptococcal cell.

PMID:37815716 | DOI:10.1007/978-1-0716-3491-2_13

Methods Mol Biol. 2024;2727:193-204. doi: 10.1007/978-1-0716-3491-2_15.

ABSTRACT

Cell wall-anchored surface proteins are integral components of Gram-positive bacterial cell envelope and vital for bacterial survival in different environmental niches. To fulfill their functions, surface protein precursors translocate from cytoplasm to bacterial cell surface in three sequential steps: secretion across the cytoplasmic membrane, covalently anchoring to the cell wall precursor lipid II by sortase A, and incorporation of the lipid II-linked precursors into mature cell wall peptidoglycan. Here, we describe a series of immunofluorescence microscopy methods to track the subcellular localization of cell wall-anchored proteins along the sorting pathway. While the protocols are tailored to Staphylococcus aureus, they can be readily adapted to localize cell wall-anchored proteins as well as membrane proteins in other Gram-positive bacteria.

PMID:37815718 | DOI:10.1007/978-1-0716-3491-2_15

Int J Pharm. 2023 Sep 22:123430. doi: 10.1016/j.ijpharm.2023.123430. Online ahead of print.

ABSTRACT

Immunotherapy has achieved some success in preclinical and clinical studies, but the immunosuppressive tumor microenvironment (TME) leads to a low response rate of this therapy. In this paper, we describe a calreticulin (CRT) valgus CT-26 tumor cell membranes-coated bacterial whole peptidoglycan (WPG) from P. aeruginosa (CPW/SR) with a high rate of the STING agonist loading. In the construct, WPG from P. aeruginosa (P.WPG) was used as a carrier with the immunoadjuvant function while synergistically promoting the maturation of dendritic cells (DCs) through the delivery of the STING agonist SR-717. CRT valgus tumor cell membranes were identified and internalized by DCs via CRT on the surface. In addition, this construct was able to reverse the immunosuppressive TME in vivo and achieve synergies with radiotherapy by creating a personalized tumor vaccine, therefore achieving more resultful antitumor efficacy. In conclusion, CPW/SR constructed in this paper provides a new approach for achieving efficient cancer immunotherapy and combination therapy.

PMID:37742823 | DOI:10.1016/j.ijpharm.2023.123430

J Pharm Biomed Anal. 2023 Oct 25;235:115627. doi: 10.1016/j.jpba.2023.115627. Epub 2023 Aug 8.

ABSTRACT

Amino acids normally have a chiral carbon and d/l-enantiomers are present. Due to the homochirality features on the present Earth, l-enantiomers are predominant in the living beings and the d-enantiomers are rare. Along with the progress and development of cutting edge analytical methods, several d-amino acids were found even in the higher animals including humans, and their biological functions and diagnostic values have also been reported. However, the amounts of these d-amino acids are much lower than the l-forms, and development/utilization of highly sensitive and selective methods are practically essential to avoid the disturbance from uncountable intrinsic substances. In the present review, multi-dimensional HPLC methods for the determination of chiral amino acids, especially two-dimensional LC-MS/MS and three-dimensional LC methods, and their applications to a variety of real-world matrices are summarized.

PMID:37633168 | DOI:10.1016/j.jpba.2023.115627

Nat Commun. 2023 Sep 2;14(1):5357. doi: 10.1038/s41467-023-41082-3.

ABSTRACT

Peptidoglycan (PG) defines cell shape and protects bacteria against osmotic stress. The growth and integrity of PG require coordinated actions between synthases that insert new PG strands and hydrolases that generate openings to allow the insertion. However, the mechanisms of their coordination remain elusive. Moenomycin that inhibits a family of PG synthases known as Class-A penicillin-binding proteins (aPBPs), collapses rod shape despite aPBPs being non-essential for rod-like morphology in the bacterium Myxococcus xanthus. Here, we demonstrate that inhibited PBP1a2, an aPBP, accelerates the degradation of cell poles by DacB, a hydrolytic PG peptidase. Moenomycin promotes the binding between DacB and PG and thus reduces the mobility of DacB through PBP1a2. Conversely, DacB also regulates the distribution and dynamics of aPBPs. Our findings clarify the action of moenomycin and suggest that disrupting the coordination between PG synthases and hydrolases could be more lethal than eliminating individual enzymes.

PMID:37660104 | PMC:PMC10475089 | DOI:10.1038/s41467-023-41082-3

bioRxiv. 2023 Aug 8:2023.08.06.552200. doi: 10.1101/2023.08.06.552200. Preprint.

ABSTRACT

Current models of bacterial cell division assume that the core synthases of the multiprotein divisome complex, FtsW-FtsI, are the primary drivers of septal peptidoglycan (PG) synthesis. These enzymes are typically encoded in the highly conserved division and cell wall ( dcw ) cluster and are considered to be universally essential for cell division. Here, we combine bioinformatics analyses with functional characterization in the pathogen Clostridioides difficile to show that dcw -encoded PG synthases have undergone a surprising specialization in the sole endospore-forming phylum, Firmicutes, to fulfill sporulation-specific roles. We describe a novel role for these enzymes in synthesizing septal PG during the sporulation-specific mode of cell division in C. difficile . Although these enzymes are directly regulated by canonical divisome components during this process, dcw -encoded PG synthases and their divisome regulators are unexpectedly dispensable for cell division during normal growth. Instead, C. difficile uses its sole bifunctional class A penicillin-binding protein (aPBP) to drive cell division, revealing a previously unreported role for this class of PG synthases as the core divisome enzyme. Collectively, our findings reveal how the emergence of endosporulation in the Firmicutes phylum was a key driver for the functional repurposing of an otherwise universally conserved cellular process such as cell division. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a divisome that differs markedly from previously studied bacteria, thus representing an attractive, unique target for therapeutic purposes.

PMID:37609260 | PMC:PMC10441361 | DOI:10.1101/2023.08.06.552200

Proc Natl Acad Sci U S A. 2023 Sep 5;120(36):e2305649120. doi: 10.1073/pnas.2305649120. Epub 2023 Aug 28.

ABSTRACT

Resilience to short-term perturbations, like inflammation, is a fundamental feature of microbiota, yet the underlying mechanisms of microbiota resilience are incompletely understood. Here, we show that Lactiplantibacillus plantarum, a major Drosophila commensal, stably colonizes the fruit fly gut during infection and is resistant to Drosophila antimicrobial peptides (AMPs). By transposon screening, we identified L. plantarum mutants sensitive to AMPs. These mutants were impaired in peptidoglycan O-acetylation or teichoic acid D-alanylation, resulting in increased negative cell surface charge and higher affinity to cationic AMPs. AMP-sensitive mutants were cleared from the gut after infection and aging-induced gut inflammation in wild-type, but not in AMP-deficient flies, suggesting that resistance to host AMPs is essential for commensal resilience in an inflamed gut environment. Thus, our work reveals that in addition to the host immune tolerance to the microbiota, commensal-encoded resilience mechanisms are necessary to maintain the stable association between host and microbiota during inflammation.

PMID:37639605 | DOI:10.1073/pnas.2305649120

bioRxiv. 2023 Aug 17:2023.08.17.553294. doi: 10.1101/2023.08.17.553294. Preprint.

ABSTRACT

Bacteria use a diverse range of carbohydrates to generate a profusion of glycans, with amino sugars such as N -acetylglucosamine (GlcNAc) being prevalent in the cell wall and in many exopolysaccharides. The primary substrate for GlcNAc-containing glycans, UDP-GlcNAc, is the product of the bacterial hexosamine pathway, and a key target for bacterial metabolic glycan engineering. Using the strategy of expressing NahK, to circumvent the hexosamine pathway, it is possible to directly feed the analogue of GlcNAc, N -azidoacetylglucosamine (GlcNAz), for metabolic labelling in E. coli . The cytosolic production of UDP-GlcNAz was confirmed using fluorescence assisted polyacrylamide gel electrophoresis. The key question of where GlcNAz is incorporated, was interrogated by analyzing potential sites including: peptidoglycan (PGN), the biofilm-related exopolysaccharide poly-β-1,6- N -acetylglucosamine (PNAG), lipopolysaccharide (LPS) and the enterobacterial common antigen (ECA). The highest levels of incorporation were observed in PGN with lower levels in PNAG and no observable incorporation in LPS or ECA. The promiscuity of the PNAG synthase (PgaCD) towards UDP-GlcNAz in vitro and lack of undecaprenyl-pyrophosphoryl-GlcNAz intermediates generated in vivo confirmed the incorporation preferences. The results of this work will guide the future development of carbohydrate-based probes and metabolic engineering strategies.

PMID:37645909 | PMC:PMC10462111 | DOI:10.1101/2023.08.17.553294