Karl Ocius

Poult Sci. 2023 Apr 13;102(7):102716. doi: 10.1016/j.psj.2023.102716. Online ahead of print.

ABSTRACT

Muramidase is an enzyme that hydrolyzes peptidoglycans of bacterial cell walls and improves performance of broilers in a dose-dependent manner. An experiment was conducted to evaluate muramidase supplementation, at a high or step-down dose, in turkeys from hatch to market. Male, B.U.T. 6 turkey poults were placed in 24 floor pens at 32 birds per pen. Poults were fed 1 of 3 diets from d 1 to 126 of age. There were 8 replicate pens per treatment. The treatments were a control (CTL) diet, the CTL plus muramidase at 45,000 LSU(F)/kg from phase 1 to 6 (BAL45), and the CTL plus muramidase at 45,000 LSU(F)/kg from phase 1 to 3 and decreased to 25,000 LSU(F)/kg from phase 4 to 6 (BAL45-25). Data were analyzed using SAS. The model included treatment and block and means were separated by Fisher LSD test. Birds fed BAL45 were heavier (P < 0.05) and had a greater (P < 0.05) average daily gain compared with birds fed the CTL from hatch to d 126 of age. Birds fed BAL45-25 had a final BW and average daily gain intermediate to or equivalent to birds fed BAL45 at the same phases. Feed conversion ratio was improved (P < 0.05) in birds fed BAL45 compared with birds fed the CTL and intermediate in birds fed BAL45-25. Breast meat yield was greater (P < 0.05) in turkeys fed muramidase, regardless of dose, compared with birds fed the CTL. There was no effect of treatment on muramic acid content in the jejunum digesta or litter scores. The frequency of pododermatitis score 1 was greater (P < 0.05) and score 2 was lower (P < 0.05) in birds fed muramidase, regardless of dose, compared with birds fed the CTL diet. In conclusion, muramidase supplementation improved performance, breast meat yield, feed efficiency and some markers of welfare, proportional to the dose in the diets.

PMID:37148570 | DOI:10.1016/j.psj.2023.102716

J Microbiol Biotechnol. 2023 Apr 20;33(7):1-9. doi: 10.4014/jmb.2302.02033. Online ahead of print.

ABSTRACT

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes was determined using the bacterial colony counting from the magnetic separations after binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as E. faecium, Staphylococcus aureus, Escherichia coli , Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

PMID:37164751 | DOI:10.4014/jmb.2302.02033

Res Sq. 2023 Apr 28:rs.3.rs-2842385. doi: 10.21203/rs.3.rs-2842385/v1. Preprint.

ABSTRACT

Objectives Peptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes. Methods Intraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response. Results A total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, with mean 8 PG occurrences per 10 mm ² of tissue in PG-positive samples. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels. Conclusion Peptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.

PMID:37162851 | PMC:PMC10168439 | DOI:10.21203/rs.3.rs-2842385/v1

ACS Chem Biol. 2023 May 12. doi: 10.1021/acschembio.3c00085. Online ahead of print.

ABSTRACT

The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N-arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N-arylpyrazole NOD2 agonist is enantiomer-specific and effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo. Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

PMID:37172210 | DOI:10.1021/acschembio.3c00085

J Biotechnol. 2023 Apr 10;367:11-19. doi: 10.1016/j.jbiotec.2023.03.007. Epub 2023 Mar 25.

ABSTRACT

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV-vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

PMID:36972749 | DOI:10.1016/j.jbiotec.2023.03.007

Nanoscale Adv. 2023 Mar 15;5(8):2251-2260. doi: 10.1039/d3na00014a. eCollection 2023 Apr 11.

ABSTRACT

Exploitation of the biotin-streptavidin interaction for advanced protein engineering is used in many bio-nanotechnology applications. As such, researchers have used diverse techniques involving chemical and enzyme reactions to conjugate biotin to biomolecules of interest for subsequent docking onto streptavidin-associated molecules. Unfortunately, the biotin-streptavidin interaction is susceptible to steric hindrance and conformational malformation, leading to random orientations that ultimately impair the function of the displayed biomolecule. To minimize steric conflicts, we employ sortase A transpeptidation to produce quantitative, seamless, and unbranched nanobody-biotin conjugates for efficient display on streptavidin-associated nanoparticles. We further characterize the protein-nanoparticle complex and demonstrate its usefulness in optical microscopy and multivalent severe acute respiratory syndrome coronavirus (SARS-CoV-2) antigen interaction. The approach reported here provides a template for making novel multivalent and multifunctional protein complexes for avidity-inspired technologies.

PMID:37056610 | PMC:PMC10089078 | DOI:10.1039/d3na00014a

Front Pharmacol. 2023 Mar 7;14:1127722. doi: 10.3389/fphar.2023.1127722. eCollection 2023.

ABSTRACT

Receptor-interacting serine/threonine kinase 2 (RIPK2) is a vital immunomodulator that plays critical roles in nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs) signaling. Stimulated NOD1 and NOD2 interact with RIPK2 and lead to the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK), followed by the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12/23. Defects in NOD/RIPK2 signaling are associated with numerous inflammatory diseases, including asthma, sarcoidosis, inflammatory bowel disease (Crohn's disease and ulcerative colitis), multiple sclerosis, and Blau syndrome. As RIPK2 is a crucial element of innate immunity, small molecules regulating RIPK2 functions are attractive to establish novel immunotherapies. The increased interest in developing RIPK2 inhibitors has led to the clinical investigations of novel drug candidates. In this review, we attempt to summarize recent advances in the development of RIPK2 inhibitors and degraders.

PMID:36959850 | PMC:PMC10028200 | DOI:10.3389/fphar.2023.1127722

Microbiol Spectr. 2023 Apr 26:e0001423. doi: 10.1128/spectrum.00014-23. Online ahead of print.

ABSTRACT

Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG dd-carboxypeptidases (DD-CPases) DacC and DacA in cell growth under alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline DD-CPase, the enzyme activity and protein stability of which are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for growth under salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all of these roles of DacC and DacA were independent of ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs)-dd-transpeptidases-mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of DD-CPases in bacterial growth and shape maintenance under stress conditions and provide novel insights into the cellular functions of DD-CPases associated with PBPs. IMPORTANCE Most bacteria have a peptidoglycan architecture for cell shape maintenance and protection against osmotic challenges. Peptidoglycan dd-carboxypeptidases control the amount of pentapeptide substrates, which are used in the formation of 4-3 cross-links by the peptidoglycan synthetic dd-transpeptidases, penicillin-binding proteins (PBPs). Seven dd-carboxypeptidases exist in Escherichia coli, but the physiological significance of their redundancy and their roles in peptidoglycan synthesis are poorly understood. Here, we showed that DacC is an alkaline dd-carboxypeptidase for which both protein stability and enzyme activity are significantly enhanced at high pH. Strikingly, dd-carboxypeptidases DacC and DacA physically interacted with PBPs, and these interactions were necessary for cell shape maintenance as well as growth under alkaline and salt stresses. Thus, cooperation between dd-carboxypeptidases and PBPs may allow E. coli to overcome various stresses and to maintain cell shape.

PMID:37098975 | DOI:10.1128/spectrum.00014-23

Angew Chem Int Ed Engl. 2023 Apr 26:e202301522. doi: 10.1002/anie.202301522. Online ahead of print.

ABSTRACT

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.

PMID:37099323 | DOI:10.1002/anie.202301522

Commun Biol. 2023 Apr 18;6(1):428. doi: 10.1038/s42003-023-04808-z.

ABSTRACT

Control of cell size and morphology is of paramount importance for bacterial fitness. In the opportunistic pathogen Enterococcus faecalis, the formation of diplococci and short cell chains facilitates innate immune evasion and dissemination in the host. Minimisation of cell chain size relies on the activity of a peptidoglycan hydrolase called AtlA, dedicated to septum cleavage. To prevent autolysis, AtlA activity is tightly controlled, both temporally and spatially. Here, we show that the restricted localization of AtlA at the septum occurs via an unexpected mechanism. We demonstrate that the C-terminal LysM domain that allows the enzyme to bind peptidoglycan is essential to target this enzyme to the septum inside the cell before its translocation across the membrane. We identify a membrane-bound cytoplasmic protein partner (called AdmA) involved in the recruitment of AtlA via its LysM domains. This work reveals a moonlighting role for LysM domains, and a mechanism evolved to restrict the subcellular localization of a potentially lethal autolysin to its site of action.

PMID:37072531 | PMC:PMC10113225 | DOI:10.1038/s42003-023-04808-z

Nat Med. 2023 Mar 13. doi: 10.1038/s41591-023-02234-6. Online ahead of print.

ABSTRACT

Increasing evidence suggests that the gut microbiome may modulate the efficacy of cancer immunotherapy. In a B cell lymphoma patient cohort from five centers in Germany and the United States (Germany, n = 66; United States, n = 106; total, n = 172), we demonstrate that wide-spectrum antibiotics treatment ('high-risk antibiotics') prior to CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is associated with adverse outcomes, but this effect is likely to be confounded by an increased pretreatment tumor burden and systemic inflammation in patients pretreated with high-risk antibiotics. To resolve this confounding effect and gain insights into antibiotics-masked microbiome signals impacting CAR-T efficacy, we focused on the high-risk antibiotics non-exposed patient population. Indeed, in these patients, significant correlations were noted between pre-CAR-T infusion Bifidobacterium longum and microbiome-encoded peptidoglycan biosynthesis, and CAR-T treatment-associated 6-month survival or lymphoma progression. Furthermore, predictive pre-CAR-T treatment microbiome-based machine learning algorithms trained on the high-risk antibiotics non-exposed German cohort and validated by the respective US cohort robustly segregated long-term responders from non-responders. Bacteroides, Ruminococcus, Eubacterium and Akkermansia were most important in determining CAR-T responsiveness, with Akkermansia also being associated with pre-infusion peripheral T cell levels in these patients. Collectively, we identify conserved microbiome features across clinical and geographical variations, which may enable cross-cohort microbiome-based predictions of outcomes in CAR-T cell immunotherapy.

PMID:36914893 | DOI:10.1038/s41591-023-02234-6

Proc Natl Acad Sci U S A. 2023 Mar 21;120(12):e2301414120. doi: 10.1073/pnas.2301414120. Epub 2023 Mar 15.

ABSTRACT

Peptidoglycan hydrolases, or autolysins, play a critical role in cell wall remodeling and degradation, facilitating bacterial growth, cell division, and cell separation. In Staphylococcus aureus, the so-called "major" autolysin, Atl, has long been associated with host adhesion; however, the molecular basis underlying this phenomenon remains understudied. To investigate, we used the type V glycopeptide antibiotic complestatin, which binds to peptidoglycan and blocks the activity of autolysins, as a chemical probe of autolysin function. We also generated a chromosomally encoded, catalytically inactive variant of the Atl enzyme. Autolysin-mediated peptidoglycan hydrolysis, in particular Atl-mediated daughter cell separation, was shown to be critical for maintaining optimal surface levels of S. aureus cell wall-anchored proteins, including the fibronectin-binding proteins (FnBPs) and protein A (Spa). As such, disrupting autolysin function reduced the affinity of S. aureus for host cell ligands, and negatively impacted early stages of bacterial colonization in a systemic model of S. aureus infection. Phenotypic studies revealed that Spa was sequestered at the septum of complestatin-treated cells, highlighting that autolysins are required to liberate Spa during cell division. In summary, we reveal the hydrolytic activities of autolysins are associated with the surface display of S. aureus cell wall-anchored proteins. We demonstrate that by blocking autolysin function, type V glycopeptide antibiotics are promising antivirulence agents for the development of strategies to control S. aureus infections.

PMID:36920922 | DOI:10.1073/pnas.2301414120

ACS Chem Biol. 2023 Mar 1. doi: 10.1021/acschembio.2c00912. Online ahead of print.

ABSTRACT

Bacteria from the genus Mycobacterium include pathogens that cause serious diseases in humans and remain as difficult infectious agents to treat. Central to these challenges are the composition and organization of the mycobacterial cell envelope, which includes unique and complex glycans. Inositol is an essential metabolite for mycobacteria due to its presence in the structural core of the immunomodulatory cell envelope glycolipids phosphatidylinositol mannoside (PIM) and PIM-anchored lipomannan (LM) and lipoarabinomannan (LAM). Despite their importance to mycobacterial physiology and pathogenesis, many aspects of PIM, LM, and LAM construction and dynamics remain poorly understood. Recently, probes that allow metabolic labeling and detection of specific mycobacterial glycans have been developed to investigate cell envelope assembly and dynamics. However, these tools have been limited to peptidoglycan, arabinogalactan, and mycolic acid-containing glycolipids. Herein, we report the development of synthetic azido inositol (InoAz) analogues as probes that can metabolically label PIMs, LM, and LAM in intact mycobacteria. Additionally, we leverage an InoAz probe to discover an inositol importer and catabolic pathway in Mycobacterium smegmatis. We anticipate that in the future, InoAz probes, in combination with bioorthogonal chemistry, will provide a valuable tool for investigating PIM, LM, and LAM biosynthesis, transport, and dynamics in diverse mycobacterial organisms.

PMID:36856664 | DOI:10.1021/acschembio.2c00912

Front Mol Biosci. 2023 Feb 15;10:1082526. doi: 10.3389/fmolb.2023.1082526. eCollection 2023.

ABSTRACT

Currently, the use of probiotic strains and their products represents a promising innovative approach as an antagonist treatment against many human diseases. Previous studies showed that a strain of Limosilactobacillus fermentum (LAC92), previously defined as Lactobacillus fermentum, exhibited a suitable amensalistic property. The present study aimed to purify the active components from LAC92 to evaluate the biological properties of soluble peptidoglycan fragments (SPFs). The cell-free supernatant (CFS) and bacterial cells were separated after 48 h of growth in MRS medium broth and treated for isolation of SPFs. Antimicrobial activity and proliferation analysis on the human cell line HTC116 were performed using technologies such as xCELLigence, count and viability, and clonogenic analysis. MALDI-MS investigation and docking analysis were performed to determine the molecular structure and hypothetical mode of action, respectively. Our results showed that the antimicrobial activity was mainly due to SPFs. Moreover, the results obtained when investigating the SPF effect on the cell line HCT116 showed substantial preliminary evidence, suggesting their significant cytostatic and quite antiproliferative properties. Although MALDI was unable to identify the molecular structure, it was subsequently revealed by analysis of the bacterial genome. The amino acid structure is called peptide 92. Furthermore, we confirmed by molecular docking studies the interaction of peptide 92 with MDM2 protein, the negative regulator of p53. This study showed that SPFs from the LAC92 strain exerted anticancer effects on the human colon cancer HCT116 cell line via antiproliferation and inducing apoptosis. These findings indicated that this probiotic strain might be a potential candidate for applications in functional products in the future. Further examination is needed to understand the specific advantages of this probiotic strain and improve its functional features to confirm these data. Moreover, deeper research on peptide 92 could increase our knowledge and help us understand if it will be possible to apply to specific diseases such as CRC.

PMID:36876040 | PMC:PMC9975264 | DOI:10.3389/fmolb.2023.1082526

Front Cell Infect Microbiol. 2023 Feb 6;13:1129043. doi: 10.3389/fcimb.2023.1129043. eCollection 2023.

ABSTRACT

BACKGROUND: Clostridium difficile infection (CDI) is common in patients with inflammatory bowel disease (IBD) and has been reported as a risk factor for poor outcome. However, gut microbiome and mycobiome of IBD patients with CDI have been barely investigated. This study aimed to assess the gut microbiome and mycobiome in IBD patients with CDI.

METHODS: We collected fecal samples from patients with active IBD and concomitant CDI (IBD-CDI group, n=25), patients with active IBD and no CDI (IBD-only group, n=51), and healthy subjects (HC, n=40). Patients' characteristics including demographic data, disease severity, and medication history were collected. Metagenomic sequencing, taxonomic and functional analysis were carried out in the samples.

RESULTS: We found that the bacterial alpha diversity of the IBD-CDI group was decreased. The bacterial and fungal beta diversity variations between IBD patients and HC were significant, regardless of CDI status. But the IBD-CDI group did not significantly cluster separately from the IBD-only group. Several bacterial taxa, including Enterococcus faecium, Ruminococcus gnavus, and Clostridium innocuum were overrepresented in the IBD-CDI group. Furthermore, IBD patients with CDI were distinguished by several fungal taxa, including overrepresentation of Saccharomyces cerevisiae. We also identified functional differences in IBD patients with CDI include enrichment of peptidoglycan biosynthesis. The network analysis indicated specific interactions between microbial markers in IBD-CDI patients.

CONCLUSION: IBD patients with CDI had pronounced microbial dysbiosis. Gut micro-ecological changes in IBD patients with CDI might provide insight into the pathological process and potential strategies for diagnosis and treatment in this subset of patients.

PMID:36814443 | PMC:PMC9940757 | DOI:10.3389/fcimb.2023.1129043

Biomolecules. 2023 Feb 15;13(2):369. doi: 10.3390/biom13020369.

ABSTRACT

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host-pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.

PMID:36830738 | PMC:PMC9953199 | DOI:10.3390/biom13020369

Front Microbiol. 2023 Feb 9;13:1089140. doi: 10.3389/fmicb.2022.1089140. eCollection 2022.

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen with increasing incidence of multidrug-resistant strains, including resistance to last-resort antibiotics, such as carbapenems. Resistances are often due to complex interplays of natural and acquired resistance mechanisms that are enhanced by its large regulatory network. This study describes the proteomic responses of two carbapenem-resistant P. aeruginosa strains of high-risk clones ST235 and ST395 to subminimal inhibitory concentrations (sub-MICs) of meropenem by identifying differentially regulated proteins and pathways. Strain CCUG 51971 carries a VIM-4 metallo-β-lactamase or 'classical' carbapenemase; strain CCUG 70744 carries no known acquired carbapenem-resistance genes and exhibits 'non-classical' carbapenem-resistance. Strains were cultivated with different sub-MICs of meropenem and analyzed, using quantitative shotgun proteomics based on tandem mass tag (TMT) isobaric labeling, nano-liquid chromatography tandem-mass spectrometry and complete genome sequences. Exposure of strains to sub-MICs of meropenem resulted in hundreds of differentially regulated proteins, including β-lactamases, proteins associated with transport, peptidoglycan metabolism, cell wall organization, and regulatory proteins. Strain CCUG 51971 showed upregulation of intrinsic β-lactamases and VIM-4 carbapenemase, while CCUG 70744 exhibited a combination of upregulated intrinsic β-lactamases, efflux pumps, penicillin-binding proteins and downregulation of porins. All components of the H1 type VI secretion system were upregulated in strain CCUG 51971. Multiple metabolic pathways were affected in both strains. Sub-MICs of meropenem cause marked changes in the proteomes of carbapenem-resistant strains of P. aeruginosa exhibiting different resistance mechanisms, involving a wide range of proteins, many uncharacterized, which might play a role in the susceptibility of P. aeruginosa to meropenem.

PMID:36845973 | PMC:PMC9948630 | DOI:10.3389/fmicb.2022.1089140

Biophys J. 2023 Feb 10;122(3S1):300a. doi: 10.1016/j.bpj.2022.11.1692.

NO ABSTRACT

PMID:36783503 | DOI:10.1016/j.bpj.2022.11.1692

Biomol Ther (Seoul). 2023 Mar 1;31(2):141-147. doi: 10.4062/biomolther.2023.008. Epub 2023 Feb 15.

ABSTRACT

Antibiotic resistance has emerged as a global threat to modern healthcare systems and has nullified many commonly used antibiotics. β-Lactam antibiotics are among the most successful and occupy approximately two-thirds of the prescription antibiotic market. They inhibit the synthesis of the peptidoglycan layer in the bacterial cell wall by mimicking the D-Ala-D-Ala in the pentapeptide crosslinking neighboring glycan chains. To date, various β-lactam antibiotics have been developed to increase the spectrum of activity and evade drug resistance. This review emphasizes the three-dimensional structural characteristics of β-lactam antibiotics regarding the overall scaffold, working mechanism, chemical diversity, and hydrolysis mechanism by β-lactamases. The structural insight into various β-lactams will provide an in-depth understanding of the antibacterial efficacy and susceptibility to drug resistance in multidrug-resistant bacteria and help to develop better β-lactam antibiotics and inhibitors.

PMID:36788654 | DOI:10.4062/biomolther.2023.008

bioRxiv. 2023 Jan 27:2023.01.26.525573. doi: 10.1101/2023.01.26.525573. Preprint.

ABSTRACT

The characterization of microbiota mechanisms in health and disease has reinvigorated pattern recognition receptors as prominent targets for immunotherapy. Notably, our recent studies on Enterococcus species revealed peptidoglycan remodeling and activation of NOD2 as key mechanisms for microbiota enhancement of immune checkpoint inhibitor therapy. Inspired by this work and other studies of NOD2 activation, we performed in silico ligand screening and developed N -arylpyrazole dipeptides as novel NOD2 agonists. Importantly, our N -arylpyrazole NOD2 agonist is enantiomer-specific, effective at promoting immune checkpoint inhibitor therapy and requires NOD2 for activity in vivo . Given the significant functions of NOD2 in innate and adaptive immunity, these next-generation agonists afford new therapeutic leads and adjuvants for a variety of NOD2-responsive diseases.

PMID:36747725 | PMC:PMC9901186 | DOI:10.1101/2023.01.26.525573

Probiotics Antimicrob Proteins. 2023 Feb 1. doi: 10.1007/s12602-023-10042-0. Online ahead of print.

ABSTRACT

Postbiotics include cell lysates (CLs), enzymes, cell wall fragments, and heat-killed bacteria derived from probiotics. Although postbiotics are increasingly being considered for their potential health-promoting properties, the effects of postbiotics on virus-mediated inflammatory responses in the intestine have not been elucidated. Hence, the present study aimed to examine whether CLs of Lactipantibacillus plantarum (LP CL) and Lacticaseibacillus rhamnosus GG (LR CL) could inhibit virus-mediated inflammatory responses in the human intestinal epithelial cell line HT-29 in vitro. Pretreatment with LP CL and LR CL significantly inhibited interleukin (IL)-8 production, which was induced by poly I:C, a synthetic analog of double-stranded RNA (dsRNA) viruses, at the mRNA and protein levels in HT-29 cells. However, peptidoglycans and heat-killed L. plantarum and L. rhamnosus GG did not effectively inhibit IL-8 production. LP CL and LR CL attenuated the poly I:C-induced phosphorylation of ERK and JNK and the activation of NF-κB, suggesting that these CLs could inhibit poly I:C-induced IL-8 production by regulating intracellular signaling pathways in HT-29 cells. Furthermore, among the short-chain fatty acids, butyrate enhanced the inhibitory effect of CLs on poly I:C-induced IL-8 production at the mRNA and protein levels in HT-29 cells, while acetate and propionate did not. Taken together, these results suggest that both LP CL and LR CL could act as potent effector molecules that can inhibit virus-mediated inflammatory responses and confer synergistic inhibitory effects with butyrate in human intestinal epithelial cells.

PMID:36720771 | DOI:10.1007/s12602-023-10042-0

PLoS Pathog. 2023 Feb 2;19(2):e1011047. doi: 10.1371/journal.ppat.1011047. Online ahead of print.

ABSTRACT

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

PMID:36730465 | DOI:10.1371/journal.ppat.1011047

Sci Adv. 2023 Jan 27;9(4):eabg6808. doi: 10.1126/sciadv.abg6808. Epub 2023 Jan 27.

ABSTRACT

Real-time localization and microbial activity information of indigenous gut microbiota over an extended period of time remains a challenge with existing visualizing methods. Here, we report a metabolic fluorine labeling (MEFLA)-based strategy for monitoring the dynamic gut microbiota via ¹⁹F magnetic resonance imaging (¹⁹F MRI). In situ labeling of different microbiota subgroups is achieved by using a panel of peptidoglycan-targeting MEFLA probes containing ¹⁹F atoms of different chemical shifts, and subsequent real-time in vivo imaging is accomplished by multiplexed hotspot ¹⁹F MRI with high sensitivity and unlimited penetration. Using this method, we realize extended visualization (>24 hours) of native gut microbes located at different intestinal sections and semiquantitative analysis of their metabolic dynamics modulated by various conditions, such as the host death and different β-lactam antibiotics. Our strategy holds great potential for noninvasive and real-time assessing of the metabolic activities and locations of the highly dynamic gut microbiota.

PMID:36706178 | DOI:10.1126/sciadv.abg6808

ISME J. 2023 Jan 23. doi: 10.1038/s41396-023-01364-6. Online ahead of print.

ABSTRACT

D-glutamate (D-Glu) is an essential component of bacterial peptidoglycans, representing an important, yet overlooked, pool of organic matter in global oceans. However, little is known on D-Glu catabolism by marine microorganisms. Here, a novel catabolic pathway for D-Glu was identified using the marine bacterium Pseudoalteromonas sp. CF6-2 as the model. Two novel enzymes (DgcN, DgcA), together with a transcriptional regulator DgcR, are crucial for D-Glu catabolism in strain CF6-2. Genetic and biochemical data confirm that DgcN is a N-acetyltransferase which catalyzes the formation of N-acetyl-D-Glu from D-Glu. DgcA is a racemase that converts N-acetyl-D-Glu to N-acetyl-L-Glu, which is further hydrolyzed to L-Glu. DgcR positively regulates the transcription of dgcN and dgcA. Structural and biochemical analyses suggested that DgcN and its homologs, which use D-Glu as the acyl receptor, represent a new group of the general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) superfamily. DgcA and DgcN occur widely in marine bacteria (particularly Rhodobacterales) and halophilic archaea (Halobacteria) and are abundant in marine and hypersaline metagenome datasets. Thus, this study reveals a novel D-Glu catabolic pathway in ecologically important marine bacteria and halophilic archaea and helps better understand the catabolism and recycling of D-Glu in these ecosystems.

PMID:36690779 | DOI:10.1038/s41396-023-01364-6

Sci Rep. 2023 Jan 20;13(1):1163. doi: 10.1038/s41598-023-27959-9.

ABSTRACT

Biofilms represent a major concern in the food industry and healthcare. The use of probiotic bacteria and their derivatives as an alternative to conventional treatments to fight biofilm development is a promising option that has provided convincing results in the last decades. Recently, membrane vesicles (MVs) produced by probiotics have generated considerable interest due to the diversity of roles they have been associated with. However, the antimicrobial activity of probiotic MVs remains to be studied. In this work, we showed that membrane vesicles produced by Lacticaseibacillus casei BL23 (LC-MVs) exhibited strong antibiofilm activity against Salmonella enterica serovar Enteritidis (S. Enteritidis) without affecting bacterial growth. Furthermore, we found that LC-MVs affected the early stages of S. Enteritidis biofilm development and prevented attachment of bacteria to polystyrene surfaces. Importantly, LC-MVs did not impact the biomass of already established biofilms. We also demonstrated that the antibiofilm activity depended on the proteins associated with the LC-MV fraction. Finally, two peptidoglycan hydrolases (PGHs) were found to be associated with the antibiofilm activity of LC-MVs. Overall, this work allowed to identify the antibiofilm properties of LC-MVs and paved the way for the use of probiotic MVs against the development of negative biofilms.

PMID:36670157 | PMC:PMC9859808 | DOI:10.1038/s41598-023-27959-9

Sci Adv. 2023 Jan 20;9(3):eadd8659. doi: 10.1126/sciadv.add8659. Epub 2023 Jan 20.

ABSTRACT

Braun's lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli, as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope-associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.

PMID:36662863 | DOI:10.1126/sciadv.add8659

Proc Natl Acad Sci U S A. 2023 Jan 24;120(4):e2209936120. doi: 10.1073/pnas.2209936120. Epub 2023 Jan 20.

ABSTRACT

Peptidoglycan, the major structural polymer forming the cell wall of bacteria, is an important mediator of physiological and behavioral effects in mammalian hosts. These effects are frequently linked to its translocation from the intestinal lumen to host tissues. However, the modality and regulation of this translocation across the gut barrier has not been precisely addressed. In this study, we characterized the absorption of peptidoglycan across the intestine and its systemic dissemination. We report that peptidoglycan has a distinct tropism for host organs when absorbed via the gut, most notably by favoring access to the brain. We demonstrate that intestinal translocation of peptidoglycan occurs through a microbiota-induced active process. This process is regulated by the parasympathetic pathway via the muscarinic acetylcholine receptors. Together, this study reveals fundamental parameters concerning the uptake of a major microbiota molecular signal from the steady-state gut.

PMID:36669110 | DOI:10.1073/pnas.2209936120