Karl Ocius

Anal Chem. 2024 May 10. doi: 10.1021/acs.analchem.4c00059. Online ahead of print.

ABSTRACT

Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work presents a field-deployable biosensor for detecting Gram-positive bacteria from exhaled breath condensates (EBCs) based on peptidoglycan recognition using an aptamer. Dielectrophoretic force is employed to enrich the bacteria in 10 s without additional equipment or steps. Concurrently, the measurement of the sensor's interfacial capacitance is coupled to quantify the bacteria during the enrichment process. By incorporation of a semiconductor condenser, the whole detection process, including EBC collection, takes about 3 min. This biosensor has a detection limit of 10 CFU/mL, a linear range of up to 10⁵ CFU/mL and a selectivity of 1479:1. It is cost-effective and disposable due to its low cost. The sensor provides a nonstaining, culture-free and PCR-independent solution for noninvasive and real-time diagnosis of Gram-positive bacterial LRTIs.

PMID:38730304 | DOI:10.1021/acs.analchem.4c00059

Anal Chem. 2024 May 10. doi: 10.1021/acs.analchem.4c00059. Online ahead of print.

ABSTRACT

Rapidly identifying and quantifying Gram-positive bacteria are crucial to diagnosing and treating bacterial lower respiratory tract infections (LRTIs). This work presents a field-deployable biosensor for detecting Gram-positive bacteria from exhaled breath condensates (EBCs) based on peptidoglycan recognition using an aptamer. Dielectrophoretic force is employed to enrich the bacteria in 10 s without additional equipment or steps. Concurrently, the measurement of the sensor's interfacial capacitance is coupled to quantify the bacteria during the enrichment process. By incorporation of a semiconductor condenser, the whole detection process, including EBC collection, takes about 3 min. This biosensor has a detection limit of 10 CFU/mL, a linear range of up to 10⁵ CFU/mL and a selectivity of 1479:1. It is cost-effective and disposable due to its low cost. The sensor provides a nonstaining, culture-free and PCR-independent solution for noninvasive and real-time diagnosis of Gram-positive bacterial LRTIs.

PMID:38730304 | DOI:10.1021/acs.analchem.4c00059

Brain Behav Immun. 2024 May 4:S0889-1591(24)00398-2. doi: 10.1016/j.bbi.2024.05.009. Online ahead of print.

ABSTRACT

Metabolites and compounds derived from gut-associated bacteria can modulate numerous physiological processes in the host, including immunity and behavior. Using a model of oral bacterial infection, we previously demonstrated that gut-derived peptidoglycan (PGN), an essential constituent of the bacterial cell envelope, influences female fruit fly egg-laying behavior by activating the NF-κB cascade in a subset of brain neurons. These findings underscore PGN as a potential mediator of communication between gut bacteria and the brain in Drosophila, prompting further investigation into its impact on all brain cells. Through high-resolution mass spectrometry, we now show that PGN fragments produced by gut bacteria can rapidly reach the central nervous system. In Addition, by employing a combination of whole-genome transcriptome analyses, comprehensive genetic assays, and reporter gene systems, we reveal that gut bacterial infection triggers a PGN dose-dependent NF-κB immune response in perineurial glia, forming the continuous outer cell layer of the blood-brain barrier. Furthermore, we demonstrate that persistent PGN-dependent NF-κB activation in perineurial glial cells correlates with a reduction in lifespan and early neurological decline. Overall, our findings establish gut-derived PGN as a critical mediator of the gut-immune-brain axis in Drosophila.

PMID:38710338 | DOI:10.1016/j.bbi.2024.05.009

Eur J Med Chem. 2024 Apr 20;271:116439. doi: 10.1016/j.ejmech.2024.116439. Online ahead of print.

ABSTRACT

Nucleotide-binding oligomerization domain 2 (NOD2) is a receptor of the innate immune system that is capable of perceiving bacterial and viral infections. Muramyl dipeptide (MDP, N-acetyl muramyl L-alanyl-d-isoglutamine), identified as the minimal immunologically active component of bacterial cell wall peptidoglycan (PGN) is recognized by NOD2. In terms of biological activities, MDP demonstrated vaccine adjuvant activity and stimulated non-specific protection against bacterial, viral, and parasitic infections and cancer. However, MDP has certain drawbacks including pyrogenicity, rapid elimination, and lack of oral bioavailability. Several detailed structure-activity relationship (SAR) studies around MDP scaffolds are being carried out to identify better NOD2 ligands. The present review elaborates a comprehensive SAR summarizing structural aspects of MDP derivatives in relation to NOD2 agonistic activity.

PMID:38691886 | DOI:10.1016/j.ejmech.2024.116439

Adv Sci (Weinh). 2024 Apr 5:e2305605. doi: 10.1002/advs.202305605. Online ahead of print.

ABSTRACT

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

PMID:38581131 | DOI:10.1002/advs.202305605

ACS Infect Dis. 2024 Apr 15. doi: 10.1021/acsinfecdis.4c00119. Online ahead of print.

ABSTRACT

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum β-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb β-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, Ldt_Mt1, Ldt_Mt2, Ldt_Mt3, and Ldt_Mt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except Ldt_Mt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (K_{I app} 9.2 ± 0.9 μM, k₂/K 5600 ± 560 M^-1 s^-1) and similar to clavulanate (K_{I app} 3.3 ± 0.6 μM, k₂/K 8400 ± 840 M^-1 s^-1); however, durlobactam had a lower turnover number (t_n = k_cat/k_inact) than clavulanate (1 and 8, respectively). K_{I app} values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and Ldt_Mt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 μg/mL, similar to the ranges for meropenem (1-32 μg/mL) and imipenem (0.5-64 μg/mL). In β-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel β-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

PMID:38619138 | DOI:10.1021/acsinfecdis.4c00119

Nat Commun. 2024 Apr 16;15(1):3286. doi: 10.1038/s41467-024-47530-y.

ABSTRACT

Food availability and usage is a major adaptive force for the successful survival of animals in nature, yet little is known about the specific signals that activate the host digestive system to allow for the consumption of varied foods. Here, by using a food digestion system in C. elegans, we discover that bacterial peptidoglycan (PGN) is a unique food signal that activates animals to digest inedible food. We identified that a glycosylated protein, Bacterial Colonization Factor-1 (BCF-1), in the gut interacts with bacterial PGN, leading to the inhibition of the mitochondrial unfolded protein response (UPR^mt) by regulating the release of Neuropeptide-Like Protein (NLP-3). Interestingly, activating UPR^mt was found to hinder food digestion, which depends on the innate immune p38 MAPK/PMK-1 pathway. Conversely, inhibiting PMK-1 was able to alleviate digestion defects in bcf-1 mutants. Furthermore, we demonstrate that animals with digestion defects experience reduced natural adaptation capabilities. This study reveals that PGN-BCF-1 interaction acts as "good-food signal" to promote food digestion and animal growth, which facilitates adaptation of the host animals by increasing ability to consume a wide range of foods in their natural environment.

PMID:38627398 | PMC:PMC11021419 | DOI:10.1038/s41467-024-47530-y

Mol Biol Cell. 2024 Apr 10:mbcE24010044. doi: 10.1091/mbc.E24-01-0044. Online ahead of print.

ABSTRACT

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light-emitting organ of its juvenile host, we showed that, after the daily dawn-triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for about 6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow-growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow-growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG-remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow-growing stage. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

PMID:38598294 | DOI:10.1091/mbc.E24-01-0044

Bioconjug Chem. 2024 Apr 17;35(4):489-498. doi: 10.1021/acs.bioconjchem.4c00007. Epub 2024 Apr 9.

ABSTRACT

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activates peptidoglycan sensors in human cells, including the nucleotide-binding oligomerization domain-containing protein 2. When present in the gastrointestinal tract, both the polymeric form (sacculi) and depolymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To utilize this growing area of biology toward therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and noninvasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a noninvasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for noninvasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.

PMID:38591251 | DOI:10.1021/acs.bioconjchem.4c00007

Infect Drug Resist. 2024 Mar 26;17:1231-1242. doi: 10.2147/IDR.S455339. eCollection 2024.

ABSTRACT

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea ZJ-2 is a novel antibacterial agent against multi-drug resistant Enterococcus faecium. In this work, we refined the bactericidal mechanism of ZJ-2 as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis.

METHODS: A wild-type strain (WT) and a mutant strain (ΔatlA) were used to investigate the effects of ZJ-2 on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia.

RESULTS: The results revealed that ZJ-2 down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (sprX, walR, atlA, and lytM), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while ΔatlA interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors.

CONCLUSION: ZJ-2 may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway.

PMID:38560705 | PMC:PMC10981453 | DOI:10.2147/IDR.S455339

Infect Immun. 2024 Apr 2:e0000424. doi: 10.1128/iai.00004-24. Online ahead of print.

ABSTRACT

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

PMID:38563734 | DOI:10.1128/iai.00004-24

Elife. 2024 Apr 5;13:e97277. doi: 10.7554/eLife.97277.

ABSTRACT

An enzyme that remodels the cell wall of Enterococcus faecium helps these gut bacteria to divide and generate peptide fragments that enhance the immune response against cancer.

PMID:38578679 | PMC:PMC10997327 | DOI:10.7554/eLife.97277

Cell Rep. 2024 Apr 6;43(4):114067. doi: 10.1016/j.celrep.2024.114067. Online ahead of print.

ABSTRACT

Mitochondrial dysfunction critically contributes to many major human diseases. The impact of specific gut microbial metabolites on mitochondrial functions of animals and the underlying mechanisms remain to be uncovered. Here, we report a profound role of bacterial peptidoglycan muropeptides in promoting mitochondrial functions in multiple mammalian models. Muropeptide addition to human intestinal epithelial cells (IECs) leads to increased oxidative respiration and ATP production and decreased oxidative stress. Strikingly, muropeptide treatment recovers mitochondrial structure and functions and inhibits several pathological phenotypes of fibroblast cells derived from patients with mitochondrial disease. In mice, muropeptides accumulate in mitochondria of IECs and promote small intestinal homeostasis and nutrient absorption by modulating energy metabolism. Muropeptides directly bind to ATP synthase, stabilize the complex, and promote its enzymatic activity in vitro, supporting the hypothesis that muropeptides promote mitochondria homeostasis at least in part by acting as ATP synthase agonists. This study reveals a potential treatment for human mitochondrial diseases.

PMID:38583150 | DOI:10.1016/j.celrep.2024.114067

Microorganisms. 2024 Feb 25;12(3):463. doi: 10.3390/microorganisms12030463.

ABSTRACT

Gut dysbiosis and subclinical intestinal damage are common in cirrhosis. The aim of this study was to examine the association of intestinal damage biomarkers (diamine oxidase [DAO], claudin 3, and intestinal fatty acid binding protein [I-FABP; FABP2]) with the state of the gut microbiota in cirrhosis. The blood levels of DAO were inversely correlated with blood levels of claudin 3, lipopolysaccharide (LPS), presepsin, TNF-α, and the severity of cirrhosis according to Child-Pugh scores. The blood level of I-FABP was directly correlated with the blood level of claudin 3 but not with that of DAO. Patients with small intestinal bacterial overgrowth (SIBO) had lower DAO levels than patients without SIBO. There was no significant difference in claudin 3 levels and I-FABP detection rates between patients with and without SIBO. The DAO level was directly correlated with the abundance of Akkermansiaceae, Akkermansia, Allisonella, Clostridiaceae, Dialister, Lactobacillus, Muribaculaceae, Negativibacillus, Ruminococcus, Thiomicrospiraceae, Verrucomicrobiae, and Verrucomicrobiota; and it was inversely correlated with the abundance of Anaerostipes, Erysipelatoclostridium, and Vibrio. The I-FABP level was directly correlated with Anaerostipes, Bacteroidia, Bacteroidota, Bilophila, Megamonas, and Selenomonadaceae; and it was inversely correlated with the abundance of Brucella, Pseudomonadaceae, Pseudomonas, and Vibrionaceae. The claudin 3 level was directly correlated with Anaerostipes abundance and was inversely correlated with the abundance of Brucella, Coriobacteriia, Eggerthellaceae, and Lactobacillus.

PMID:38543514 | PMC:PMC10972037 | DOI:10.3390/microorganisms12030463

Immunohorizons. 2024 Mar 1;8(3):269-280. doi: 10.4049/immunohorizons.2300109.

ABSTRACT

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36, and TIM-3, whereas TIM-1, αVβ3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

PMID:38517345 | DOI:10.4049/immunohorizons.2300109