Karl Ocius

Rheumatology (Oxford). 2024 Sep 9:keae476. doi: 10.1093/rheumatology/keae476. Online ahead of print.

ABSTRACT

OBJECTIVE: Bacterial translocation across the gut barrier has been implicated in the pathogenesis of systemic lupus erythematosus (SLE), though underlying mechanisms remain unclear. This study aimed to investigate the role of translocated bacteria in the context of molecular mimicry by utilizing lupus model mice and blood samples from untreated SLE patients.

METHODS: Bacterial translocation was evaluated using nonselective cultured mesenteric lymph nodes (MLNs) from B6SKG mice, a lupus model characterized by impaired TCR signalling and gut dysbiosis. The relationships of detected pathobionts with autoantibody production were examined using in vivo experiments, enzyme-linked immunosorbent assay, immunoblotting, and epitope mapping.

RESULTS: Culture-based bacterial profiling in MLNs demonstrated that Lactobacillus murinus was enriched in B6SKG mice with elevated anti-dsDNA IgG levels. Subcutaneous injection of heat-killed L. murinus induced anti-dsDNA IgG production without altering T- or B cell subset composition. Immunoblotting and mass spectrometry analysis identified a peptide ATP-binding cassette (ABC) transporter as a molecular mimicry antigen, with its cross-reactivity in lupus mice confirmed by serological assays and in vivo immunization. The L. murinus ABC transporter exhibited surface epitopes that were cross-reactive with sera from lupus mice and patients. The ABC transporter from R. gnavus, known for its pathogenic role in lupus patients, had a similar epitope sequence to that of the L. murinus ABC transporter and reacted with lupus sera.

CONCLUSION: ABC transporters from gut bacteria can serve as cross-reactive antigens that may promote anti-dsDNA antibody production in genetically susceptible mice. These findings underscore the role of commensal-derived molecular mimicry and bacterial translocation in lupus pathogenesis.

PMID:39250733 | DOI:10.1093/rheumatology/keae476

Chem Biol Drug Des. 2024 Sep;104(3):e14612. doi: 10.1111/cbdd.14612.

ABSTRACT

The Mycobacterium cell wall is a capsule-like structure comprising of various layers of biomolecules such as mycolic acid, peptidoglycans, and arabinogalactans, which provide the Mycobacteria a sort of cellular shield. Drugs like isoniazid, ethambutol, cycloserine, delamanid, and pretomanid inhibit cell wall synthesis by inhibiting one or the other enzymes involved in cell wall synthesis. Many enzymes present across these layers serve as potential targets for the design and development of newer anti-TB drugs. Some of these targets are currently being exploited as the most druggable targets like DprE1, InhA, and MmpL3. Many of the anti-TB agents present in clinical trials inhibit cell wall synthesis. The present article covers a systematic perspective of developing cell wall inhibitors targeting various enzymes involved in cell wall biosynthesis as potential drug candidates for treating Mtb infection.

PMID:39237482 | DOI:10.1111/cbdd.14612

Bioconjug Chem. 2024 Sep 18;35(9):1335-1342. doi: 10.1021/acs.bioconjchem.4c00264. Epub 2024 Aug 22.

ABSTRACT

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (¹⁸F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific ¹⁸F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[¹⁸F]FH₃RESCA)-NH₂) or the unlabeled chelator (H-GGGK((±)-H₃RESCA)-NH₂) followed by labeling with Al[¹⁸F]F, respectively. The overall radiochemical yields were 15-43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% (n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable ¹⁸F-labeling method to prepare nanobodies (Nb-Al[¹⁸F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

PMID:39172920 | DOI:10.1021/acs.bioconjchem.4c00264

J Bacteriol. 2024 Sep 25:e0020424. doi: 10.1128/jb.00204-24. Online ahead of print.

ABSTRACT

Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 is responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation and in which the connection between Wag31, PlrA, and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate the synthesis of the different layers of the mycobacterial cell wall in changing conditions.

IMPORTANCE: This study is performed in Mycobacterium smegmatis, which is used as a model to understand the basic physiology of pathogenic mycobacteria such as Mycobacterium tuberculosis. In this work, we examine the function and regulation of three proteins involved in regulating cell wall elongation in mycobacterial cells, which occurs at the cell tips or poles. We find that Wag31, a regulator of polar elongation, works partly through the regulation of MmpL3, a transporter of cell wall constituents and an important drug target. Our work suggests that, beyond its transport function, MmpL3 has another function in controlling cell wall synthesis broadly in response to stress.

PMID:39320104 | DOI:10.1128/jb.00204-24

Nat Commun. 2024 Sep 19;15(1):8225. doi: 10.1038/s41467-024-52570-5.

NO ABSTRACT

PMID:39300107 | PMC:PMC11413288 | DOI:10.1038/s41467-024-52570-5

Molecules. 2024 Aug 27;29(17):4065. doi: 10.3390/molecules29174065.

ABSTRACT

The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.

PMID:39274911 | PMC:PMC11396672 | DOI:10.3390/molecules29174065

Acta Pharm. 2024 Sep 14;74(3):423-440. doi: 10.2478/acph-2024-0024. Print 2024 Sep 1.

ABSTRACT

In the final phases of bacterial cell wall synthesis, penicillin-binding proteins (PBPs) catalyze the cross-linking of peptidoglycan. For many decades, effective and non-toxic β-lactam antibiotics have been successfully used as mimetics of the d-Ala-d-Ala moiety of the natural substrate and employed as irreversible inhibitors of PBPs. In the years following their discovery, the emergence of resistant bacteria led to a decline in their clinical efficacy. Using Staudinger cycloaddition, we synthesized a focused library of novel monocyclic β-lactams in which different substituents were introduced at the C4 position of the β-lactam ring, at the C3 amino position, and at the N1 lactam nitrogen. In biochemical assays, the compounds were evaluated for their inhibitory effect on the model enzyme PBP1b from Streptococcus pneumoniae. Upon investigation of the antibacterial activity of the newly prepared compounds against ESKAPE pathogens, some compounds showed moderate inhibition. We also examined their reactivity and selectivity in a biochemical assay with other enzymes that have a catalytic serine in the active site, such as human cholinesterases, where they also showed no inhibitory activity, highlighting their specificity for bacterial targets. These compounds form the basis for further work on new monocyclic β-lactams with improved antibacterial activity.

PMID:39279527 | DOI:10.2478/acph-2024-0024

Chem Biol Drug Des. 2024 Sep;104(3):e14612. doi: 10.1111/cbdd.14612.

ABSTRACT

The Mycobacterium cell wall is a capsule-like structure comprising of various layers of biomolecules such as mycolic acid, peptidoglycans, and arabinogalactans, which provide the Mycobacteria a sort of cellular shield. Drugs like isoniazid, ethambutol, cycloserine, delamanid, and pretomanid inhibit cell wall synthesis by inhibiting one or the other enzymes involved in cell wall synthesis. Many enzymes present across these layers serve as potential targets for the design and development of newer anti-TB drugs. Some of these targets are currently being exploited as the most druggable targets like DprE1, InhA, and MmpL3. Many of the anti-TB agents present in clinical trials inhibit cell wall synthesis. The present article covers a systematic perspective of developing cell wall inhibitors targeting various enzymes involved in cell wall biosynthesis as potential drug candidates for treating Mtb infection.

PMID:39237482 | DOI:10.1111/cbdd.14612

Gut Microbes. 2024 Jan-Dec;16(1):2395099. doi: 10.1080/19490976.2024.2395099. Epub 2024 Sep 6.

ABSTRACT

The intestinal microbiota of humans includes a highly diverse range of bacterial species. All these bacteria possess a cell wall, composed primarily of the macromolecule peptidoglycan. As such, the gut also harbors an abundant and varied peptidoglycome. A remarkable range of host physiological pathways are regulated by peptidoglycan fragments that originate from the gut microbiota and enter the host system. Interactions between the host system and peptidoglycan can influence physiological development and homeostasis, promote health, or contribute to inflammatory disease. Underlying these effects is the interplay between microbiota composition and enzymatic processes that shape the intestinal peptidoglycome, dictating the types of peptidoglycan generated, that subsequently cross the gut barrier. In this review, we highlight and discuss the hidden and emerging functional aspects of the microbiome, i.e. the hidden base of the iceberg, that modulate the composition of gut peptidoglycan, and how these fundamental processes are drivers of physiological outcomes for the host.

PMID:39239828 | PMC:PMC11382707 | DOI:10.1080/19490976.2024.2395099

Anal Methods. 2024 Aug 27. doi: 10.1039/d4ay00982g. Online ahead of print.

ABSTRACT

The growing awareness of the health benefits associated with probiotics has led to an increasing interest in probiotic products. To develop probiotic functional foods that deliver health benefits, it is essential to characterize both probiotic viability (the ability to survive) and vitality (the ability to remain active and effective). However, traditional probiotic assays only provide limited information about their survival state. To gain a comprehensive understanding of probiotic states, a D-amino-acid-based metabolic labeling strategy was applied to quantitatively depict probiotic vitality. In this approach, probiotics were first metabolically incorporated with azido-modified D-lysine and then labeled with dibenzocyclooctyne-sulfo-Cy5 through click chemistry. This two-step labeling process provides a visual representation of the metabolic levels of probiotics as well as the bacterial membrane integrity. Besides, this method is capable of characterizing the influence of various environmental conditions, from manufacturing to oral administration, on probiotic vitality. With its rapid detection process and general applicability, this strategy has the potential to be widely implemented in the food industry for probiotic vitality evaluation.

PMID:39189146 | DOI:10.1039/d4ay00982g

Nat Commun. 2024 Aug 20;15(1):7124. doi: 10.1038/s41467-024-51067-5.

ABSTRACT

Point-of-care serological and direct antigen testing offers actionable insights for diagnosing challenging illnesses, empowering distributed health systems. Here, we report a POC-compatible serologic test for Lyme disease (LD), leveraging synthetic peptides specific to LD antibodies and a paper-based platform for rapid, and cost-effective diagnosis. Antigenic epitopes conserved across Borrelia burgdorferi genospecies, targeted by IgG and IgM antibodies, are selected to develop a multiplexed panel for detection of LD antibodies from patient sera. Multiple peptide epitopes, when combined synergistically with a machine learning-based diagnostic model achieve high sensitivity without sacrificing specificity. Blinded validation with 15 LD-positive and 15 negative samples shows 95.5% sensitivity and 100% specificity. Blind testing with the CDC's LD repository samples confirms the test accuracy, matching lab-based two-tier results, correctly differentiating between LD and look-alike diseases. This LD diagnostic test could potentially replace the cumbersome two-tier testing, improving diagnosis and enabling earlier treatment while facilitating immune monitoring and surveillance.

PMID:39164226 | PMC:PMC11336255 | DOI:10.1038/s41467-024-51067-5

Adv Mater. 2024 Oct;36(40):e2407189. doi: 10.1002/adma.202407189. Epub 2024 Aug 22.

ABSTRACT

Hematological malignancies (HM) like acute myeloid leukemia (AML) are often intractable. Cancer vaccines possibly inducing robust and broad anti-tumor immune responses may be a promising treatment option for HM. Few effective vaccines against blood cancers are, however, developed to date partly owing to insufficient stimulation of dendritic cells (DCs) in the body and lacking appropriate tumor antigens (Ags). Here it is found that systemic multifunctional nanovaccines consisting of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 9 (TLR9) agonists - muramyl dipeptide (MDP) and CpG, and tumor cell lysate (TCL) as Ags (MCA-NV) induce potent and broad immunity against AML. MCA-NV show complementary stimulation of DCs and prime homing to lymphoid organs following systemic administration. Of note, in orthotopic AML mouse models, intravenous infusion of different vaccine formulations elicits substantially higher anti-AML efficacies than subcutaneous administration. Systemic MCA-NV cure 78% of AML mice and elicit long-term immune memory with 100% protection from rechallenging AML cells. Systemic MCA-NV can also serve as prophylactic vaccines against the same AML. These systemic nanovaccines utilizing patient TCL as Ags and dual adjuvants to elicit strong, durable, and broad immune responses can provide a personalized immunotherapeutic strategy against AML and other HM.

PMID:39171954 | DOI:10.1002/adma.202407189

Anal Chem. 2024 Aug 14. doi: 10.1021/acs.analchem.4c02862. Online ahead of print.

ABSTRACT

Monitoring the effector function of cytotoxic T lymphocytes (CTLs) in vivo remains a great challenge. Here, we develop a chemistry-enabled enzymatic labeling approach to evaluate the tumor-specific immune response of CTLs by precisely monitoring the interaction between CTLs and tumor cells. Staphylococcus aureus sortase A (SrtA) is linked to the CTL surface through bioconjugate chemistry and then catalyzes the transfer of fluorescent-labeled substrate, 5-Tamra-LPETG, to CTLs. Meanwhile, the tumor cells are specifically decorated with N-terminal glycine residues (G₅ peptide) through the inherent glycolmetabolism of cathepsin B-specific cleavable triacetylated N-azidoacetyl-d-mannosamine (CB-Ac₃ManNAz) and click chemistry. After the infiltration of engineered CTLs into the tumor tissues, the immune-synapse-mediated specific interaction of CTLs and tumor cells leads to the accurate fluorescent labeling of tumor cells through the SrtA-catalyzed 5-Tamra-LPETG transfer. Therefore, the immune effect of CTLs as well as the performance of immune drugs can be determined, providing a novel strategy for pushing ahead immunotherapy.

PMID:39140208 | DOI:10.1021/acs.analchem.4c02862

Proc Natl Acad Sci U S A. 2024 Aug 20;121(34):e2408540121. doi: 10.1073/pnas.2408540121. Epub 2024 Aug 16.

ABSTRACT

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

PMID:39150786 | PMC:PMC11348318 | DOI:10.1073/pnas.2408540121