Karl Ocius

ACS Infect Dis. 2024 Dec 4. doi: 10.1021/acsinfecdis.4c00370. Online ahead of print.

ABSTRACT

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC₅₀ values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, k_inact/K_I, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, k_inact/K_I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell k_inact/K_I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro k_inact/K_I data and show a comparable relationship to previously established IC₅₀ values. These results support the validity of our in vivo k_inact/K_I method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

PMID:39628314 | DOI:10.1021/acsinfecdis.4c00370

J Med Chem. 2024 Dec 5. doi: 10.1021/acs.jmedchem.4c02139. Online ahead of print.

ABSTRACT

Helicobacter pylori (H. pylori) infection is characterized by the complex interplay between H. pylori and gastric disorders. It has been established that NOD1 can be activated by the peptidoglycan (PGN) present in the cell wall of H. pylori, serving as a key mediator of inflammation and initiating the RIP2/NF-κB and MAPK inflammatory signaling pathways. In this article, we reported on the development of a 2-chloroquinazolin-4-ol derivative 66 as a potent and selective antagonist of both human and mouse NOD1, which effectively inhibited the expression of inflammatory cytokines (IL-6, TNF-α) and chemokines (CXCL1, CXCL8) in immune and epithelial cells, as well as inflammatory cytokines (KC, IL-6) in a H. pylori-induced murine model of gastritis following oral administration. This study laid a foundation for treating gastritis induced by H. pylori infection.

PMID:39637404 | DOI:10.1021/acs.jmedchem.4c02139

Anal Chem. 2024 Nov 15. doi: 10.1021/acs.analchem.4c02851. Online ahead of print.

ABSTRACT

We developed a new method for stable carbon and nitrogen isotopic (δ¹³C and δ¹⁵N) analysis of underivatized amino acid (AA) enantiomers simultaneously, based on high-performance liquid chromatography (HPLC) separation and off-line isotopic measurement. l- and d-Enantiomers of each AA were isolated using a ReproSil Chiral-AA column, purified by wet chemical procedure, and analyzed for δ¹³C and δ¹⁵N values with a nanomol-scale elemental analyzer/isotope-ratio mass spectrometry (nano-EA/IRMS) system. We successfully achieved the separation of l- and d-enantiomers of 15 proteinogenous AAs, with all l-enantiomers eluting before respective d-enantiomers. The δ¹³C and δ¹⁵N values of AA enantiomers were consistent before and after HPLC separation, demonstrating that this analytical method conserves isotopic information. By coupling this column with a multidimensional HPLC system for isolating individual AAs, we analyzed l- and d-AAs in a natural sample, peptidoglycan isolated from Gram-positive bacterium Bacillus subtilis. Results show a surprisingly large ¹⁵N-depletion, up to 20‰, in d-glutamic acid relative to its l-counterpart. The first example, to our knowledge, of δ¹³C and δ¹⁵N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.

PMID:39546634 | DOI:10.1021/acs.analchem.4c02851

Microb Pathog. 2024 Nov 7;198:107074. doi: 10.1016/j.micpath.2024.107074. Online ahead of print.

ABSTRACT

Tuberculosis continues to pose a health challenge causing the loss of millions of lives despite the existence of multiple drugs, for treatment. The emergence of drug-resistant strains has made the situation more complex making it increasingly difficult to fight against this disease. This review outlines the challenges associated with TB drug discovery, the nature of Mycobacterium tuberculosis shedding light on the mechanisms that lead to treatment failure and antibiotic resistance. We explore promising drug targets, encompassing inhibition of mycolyarabinogalactan peptidoglycan (MAGP) assembly, mycolic acid biosynthesis, DNA replication, transcription, translation, protein synthesis, and bioenergetics/metabolism pathways. A comprehensive overview of the global pipeline of anti-tuberculosis drugs at various stages of clinical trials, the diverse strategies being pursued to tackle this complex disease. By gaining an understanding of the mechanisms that contribute to resistance development and identifying suitable targets, we can pave the way for more effective treatments and contribute to global efforts to combat drug-resistant tuberculosis.

PMID:39521155 | DOI:10.1016/j.micpath.2024.107074

iScience. 2024 Oct 11;27(11):111130. doi: 10.1016/j.isci.2024.111130. eCollection 2024 Nov 15.

ABSTRACT

Obesity is associated with compartmentalized changes in immune responses that can be protective or pathogenic. It has been proposed that obesity-related changes in the microbiota influence allergic lung inflammation. We hypothesized that sensors of the bacterial cell wall influenced allergenic lung inflammation during obesity. Ovalbumin (OVA)-induced lung inflammation was similar in female Nod1^-/- and wild-type mice during high-fat-diet-induced obesity, but allergic lung inflammation was higher in obese, high-fat-diet-fed female Nod2^-/- mice. Obese Nod2^-/- mice had higher inflammatory cell infiltration in the bronchial alveolar lavage (BAL) and lungs, pulmonary fibrosis, mucus levels, hypertrophy and hyperplasia of goblet cells, M2 alveolar macrophage infiltration, interleukin-4 (IL-4), IL-5, IL-6, and lower CXCL1 and IL-22. Therefore, Nod2 protects against excessive lung inflammation and is a bacterial sensor that relays protective responses to allergenic lung inflammation in obese female mice.

PMID:39507249 | PMC:PMC11539594 | DOI:10.1016/j.isci.2024.111130

J Med Chem. 2024 Nov 6. doi: 10.1021/acs.jmedchem.4c02196. Online ahead of print.

ABSTRACT

Vancomycin and norvancomycin have diminished antibacterial efficacy due to acquired or intrinsic resistance from mutations in the terminal dipeptide of lipid II in Gram-positive bacteria or failure to penetrate into the periplasm in Gram-negative bacteria. Herein, we rationally designed and synthesized a series of vancomycin analogues bearing single amine or guanidine functionality, altering various linkers and modification sites, to combat the resistance. Extensive antibacterial screening was performed to delineate a comprehensive SAR. Many derivatives revitalized the activity in vitro, exhibiting a 4-128-fold or 2-16-fold enhancement against the acquired or intrinsic resistance with lower toxicity. Significantly, the optimal compound 4g demonstrated greater pharmacokinetic and pharmacodynamic profiles. Further studies uncovered additional independent and synergistic mechanisms for 4g, including the enhanced membrane activity and augmented inhibition of peptidoglycan biosynthesis via increased lipid II binding, highlighting its potential as a future lead candidate to replenish the glycopeptide antibiotic arsenal.

PMID:39504470 | DOI:10.1021/acs.jmedchem.4c02196

Sci Adv. 2024 Oct 4;10(40):eadp8266. doi: 10.1126/sciadv.adp8266. Epub 2024 Oct 2.

ABSTRACT

The efficacy of immunotherapy targeting the PD-1/PD-L1 pathway in hepatocellular carcinoma (HCC) is limited. NOD-like receptors (NLRs) comprise a highly evolutionarily conserved family of cytosolic bacterial sensors, yet their impact on antitumor immunity against HCC remains unclear. In this study, we uncovered that NOD1, a well-studied member of NLR family, exhibits predominant expression in tumor-associated macrophages (TAMs) and correlates positively with improved prognosis and responses to anti-PD-1 treatments in patients with HCC. Activation of NOD1 in vivo augments antitumor immunity and enhances the effectiveness of anti-PD-1 therapy. Mechanistically, NOD1 activation resulted in diminished expression of perilipin 5, thereby hindering fatty acid oxidation and inducing free fatty acid accumulation in TAMs. This metabolic alteration promoted membrane localization of the costimulatory molecule OX40L in a lipid modification-dependent manner, thereby activating CD8⁺ T cells. These findings unveil a previously unrecognized role for NOD1 in fortifying antitumor T cell immunity in HCC, potentially advancing cancer immunotherapy.

PMID:39356756 | PMC:PMC11446285 | DOI:10.1126/sciadv.adp8266

Front Immunol. 2024 Oct 22;15:1442095. doi: 10.3389/fimmu.2024.1442095. eCollection 2024.

ABSTRACT

The mammalian gastrointestinal tract quickly becomes densely populated with foreign microorganisms shortly after birth, thereby establishing a lifelong presence of a microbial community. These commensal gut microbiota serve various functions, such as providing nutrients, processing ingested compounds, maintaining gut homeostasis, and shaping the intestinal structure in the host. Dysbiosis, which is characterized by an imbalance in the microbial community, is closely linked to numerous human ailments and has recently emerged as a key factor in health prognosis. Tuberculosis (TB), a highly contagious and potentially fatal disease, presents a pressing need for improved methods of prevention, diagnosis, and treatment strategies. Thus, we aim to explore the latest developments on how the host's immune defenses, inflammatory responses, metabolic pathways, and nutritional status collectively impact the host's susceptibility to or resilience against Mycobacterium tuberculosis infection. The review addresses how the fluctuations in the gut microbiota not only affect the equilibrium of these physiological processes but also indirectly influence the host's capacity to resist M. tuberculosis. This work highlights the central role of the gut microbiota in the host-microbe interactions and provides novel insights for the advancement of preventative and therapeutic approaches against tuberculosis.

PMID:39502685 | PMC:PMC11534664 | DOI:10.3389/fimmu.2024.1442095

Elife. 2024 Nov 4;13:RP93673. doi: 10.7554/eLife.93673.

ABSTRACT

Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against Staphylococcus aureus whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined. In this work, we have employed NMR spectroscopy to study both the substrate specificity and the bond cleavage of the bactericide lysostaphin and the S. aureus PG hydrolase LytM. Yet, we provide substrate-level evidence for the functional role of these enzymes. Indeed, our results show that the substrate specificities of these structurally highly homologous enzymes are similar, but unlike observed earlier both LytM and lysostaphin prefer the D-Ala-Gly cross-linked part of mature peptidoglycan. However, we show that while lysostaphin is genuinely a glycyl-glycine hydrolase, LytM can also act as a D-alanyl-glycine endopeptidase.

PMID:39495121 | PMC:PMC11534333 | DOI:10.7554/eLife.93673

Res Sq [Preprint]. 2024 Oct 14:rs.3.rs-5118704. doi: 10.21203/rs.3.rs-5118704/v1.

ABSTRACT

Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are implicated in the control of the intestinal microbiota; however, molecular requirements for peptidoglycan (PGN) binding and receptor signaling mechanisms remain poorly understood. We identified PGLYRP-1 as a receptor for the disaccharide motif of lysine N-acetylglucosamine N-acetylmuramic tripeptide (GMTriP-K) with a newly constructed PGN microarray. Surprisingly, PGLYRP-1 was required for innate immune activation of macrophages by GMTriP-K but not N-acetylglucosamine N-acetylmuramic dipeptide (GMDiP) or muramyl dipeptide (MDP). In macrophages, intracellular PGLYRP-1 complexed with NOD2 and GEF-H1, both of which were required for GMTriP-K-regulated gene expression. PGLYRP-1 localized to the endoplasmic reticulum and interacted at the Golgi with NOD2 upon GMTriP-K stimulation. PGLYRP-1 upregulation and its dependent gene expression signatures were induced in both mouse intestinal inflammation and human ulcerative colitis. Importantly, PGLYRP-1 activation by GMTriP-K resulted in innate immune activation and protection of mice from colitis. Our results show that PGLYRPs can function as intracellular PGN pattern recognition receptors for the control of host defense responses in the intestine.

PMID:39483916 | PMC:PMC11527351 | DOI:10.21203/rs.3.rs-5118704/v1

Liver Int. 2024 Oct 29. doi: 10.1111/liv.16143. Online ahead of print.

ABSTRACT

BACKGROUND AND AIMS: NOD2 mutations are associated with impaired gut mucosal barrier function. According to the systemic inflammation hypothesis, bacterial translocation is central in the development of decompensation. The aim was to evaluate whether the presence of NOD2 variants is associated with the development of first decompensation.

METHOD: Secondary analysis of prospectively collected consecutive patients with compensated cirrhosis, who were screened between 2014 and 2018. Patients with and without NOD2 variants were compared and stratified analysis according to the presence of varices was performed.

RESULTS: 360 patients [239 (66%) men, median age 61 (53-69) years, 70 (19%) with NOD2 variants, 90 (25%) with varices] were followed for a median of 9 (4-16) months. Similar baseline characteristics were observed across NOD2 status groups, except for beta-blocker use (45% vs. 32% amongst variant carriers vs. non-carriers, p = 0.05). During follow-up, 34 patients (12%) developed their first decompensation, with no differences according to NOD2 status [HR 1.75 (95% CI 0.84-3.67)]. On multivariate analysis, only MELD remained an independent predictor of decompensation. Amongst patients with varices (n = 90), 18 (24.4%) carried a NOD2 variants, with a higher incidence of first decompensation [HR 3.00 (95% CI 1.08-8.32)], primarily due to ascites [HR 3.32 (95% CI 1.07-10.32)]. In this subgroup, MELD [HR 1.18 (95% CI 1.06-1.32)] and NOD2 variants [HR 2.91 (95% CI 0.95-8.89)] were determined to be independent predictors of decompensation.

CONCLUSIONS: The presence of NOD2 risk variants leads to a greater incidence of first decompensation only in compensated patients with varices.

PMID:39469976 | DOI:10.1111/liv.16143