Biology subjects

Dobos, K. M.

Publications and source records attributed to Dobos, K. M..

5 recordsLinked to original sources

bioRxiv2025

Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

The antigen presenting molecule MHC class I-related protein 1 (MR1) binds small molecule metabolites derived from microbial riboflavin biosynthetic pathways and presents them at the cell surface for surveillance by MR1-restricted mucosal-associated invariant T cells (MAIT cells). MR1 ligands can originate in the extracellular space or in endosomal compartments that contain microbial pathogens. Distinct, complementary antigen processing and presentation pathways enable MR1 to survey diverse intracellular locations and present both exogenous and intracellular antigens. Here, we generated a panel of BEAS-2B MR1 KO cells reconstituted with MR1 proteins mutated at amino acids 9 - 16. The mutated MR1 molecules differentially translocated to the cell surface in response to 6-formylpterin and differed in their ability to present mycobacterial antigens to MAIT cell clones. While they barely presented Mycobacterium smegmatis supernatant and other exogenous MAIT cell antigens, their ability to present antigens derived from mycobacterial infection and a 5-A-RU prodrug requiring endosomal processing remained largely intact. Protein co-immunoprecipitation and mass spectrometry-based proteomic analysis showed that mutated MR1 differentially associated with calnexin and {beta}2-microglobulin (B2M). Knock-down of B2M in cells over-expressing MR1 phenocopied the loss of exogenous antigen presentation but did not impact presentation of intracellular antigens. Thus, the MR1-mediated presentation of exogenous antigen appears to be limited by binding to B2M whereas the lower sensitivity to B2M deficiency implies that MAIT cell activation via the endosomal antigen presentation pathway may be limited by the availability of MR1 itself.

immunology↗

bioRxiv2024

Disruption of riboflavin biosynthesis in mycobacteria establishes 5-amino-6-D-ribitylaminouracil (5-A-RU) as key precursor of MAIT cell agonists

Mucosal-associated invariant T (MAIT) cells exhibit an intrinsic ability to recognize and respond to microbial infections. The semi-invariant antigen recognition receptor of MAIT cells specifically detects the non-polymorphic antigen-presenting molecule, major histocompatibility complex class I-related protein 1 (MR1), which primarily binds riboflavin-derived metabolites of microbial origin. To further interrogate the dependence of these antigens on riboflavin biosynthesis in mycobacteria, we deleted individual genes in the riboflavin biosynthesis pathways in Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb) and evaluated the impact thereof on MAIT cell activation. Blocking the early steps of the pathway by deletion of RibA2 or RibG profoundly reduced, but did not completely ablate, MAIT cell activation by Msm or Mtb, whereas deletion of RibC, which catalyzes the last step in the pathway, had no significant effect. Interestingly, deletion of RibH specifically enhanced MAIT cell recognition of Mtb whereas loss of lumazine synthase (RibH) activity had no impact on MAIT cell activation by Msm. MAIT cell activation by Msm was likewise unaffected by blocking the production of the MAIT cell antagonist, Fo (by inhibiting its conversion from the riboflavin pathway intermediate, 5-amino-6-D-ribitylaminouracil (5-A-RU), through the deletion of fbiC). Together, these results confirm a central role for 5-AR-U in generating mycobacterial MR1 ligands and reveal similarities and differences between Msm and Mtb in terms of the impact of riboflavin pathway disruption on MAIT cell activation. Author summaryMucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T-cells which respond to microbial infections. These specialized cells recognize the MR1 molecule, which presents microbial metabolites derived from riboflavin (vitamin B2) biosynthesis. These cells are enriched in the airways and in some cases reduced in the peripheral blood of tuberculosis (TB) infected individuals suggestive of a role in the early response to infection by Mycobacterium tuberculosis. In this study, we investigated the effect of deleting individual genes in the riboflavin biosynthesis pathway on MAIT cell activation by Mycobacterium tuberculosis or Mycobacterium smegmatis. Our findings revealed that disrupting early stages in the pathway profoundly reduced but did not eliminate MAIT cell activation by both mycobacterial species. However, blocking the penultimate step in the pathway, catalyzed by the enzyme, lumazine synthase, led specifically to increased MAIT cell recognition of M. tuberculosis. Our results confirm the pivotal role of the riboflavin pathway intermediate, 5-A-RU, in generating mycobacterial ligands that serve as MAIT cell agonists. By enhancing our understanding of how MAIT cells recognize mycobacterial infections, the results of this study could inform strategies for the development of vaccines and/or immunotherapies for TB.

immunology↗

bioRxiv2022

Deaza-modification of MR1 ligands modulates recognition by MR1-restricted T cells

MR1-restricted T (MR1T) cells recognize microbial small molecule metabolites presented on the MHC Class I-like molecule MR1 and have been implicated in early effector responses to microbial infection. As a result, there is considerable interest in identifying chemical properties of metabolite ligands that permit recognition by MR1T cells, for consideration in therapeutic or vaccine applications. Here, we made chemical modifications to known MR1 ligands to evaluate the effect on MR1T cell activation. Specifically, we modified 6,7-dimethyl-8-D-ribityllumazine (DMRL) to generate 6,7-dimethyl-8-D-ribityldeazalumazine (DZ), and then further derivatized DZ to determine the requirements for retaining MR1 surface stabilization and agonistic properties. Interestingly, the IFN-{gamma} response toward DZ varied widely across a panel of T cell receptor (TCR)-diverse MR1T cell clones; while one clone was agnostic toward the modification, most displayed either an enhancement or depletion of IFN-{gamma} production when compared with its response to DMRL. To gain insight into a putative mechanism behind this phenomenon, we used in silico molecular docking techniques for DMRL and its derivatives and performed molecular dynamics simulations of the complexes. In assessing the dynamics of each ligand in the MR1 pocket, we found that DMRL and DZ exhibit differential dynamics of both the ribityl moiety and the aromatic backbone, which may contribute to ligand recognition. Together, our results support an emerging hypothesis for flexibility in MR1:ligand-MR1T TCR interactions and enable further exploration of the relationship between MR1:ligand structures and MR1T cell recognition for downstream applications targeting MR1T cells.

immunology↗

bioRxiv2022

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

microbiology↗

bioRxiv2021

A Dose-Response Model and D10-Value for Mycobacterium tuberculosis Exposed to Dosimetrically Verified Ionizing Radiation

Techniques for pathogen inactivation have been employed by laboratories to help ease the financial, physical, and health strains associated with (A)BSL-3 work. Exposure to radiation is the most common and useful of these methods to inactivate pathogens grown in large-scale culture. While robust protocols exist for radiation exposure techniques, there are variances in methods used to determine the radiation dose and dose rate required to inactivate pathogens. Furthermore, previous studies often do not include radiation dosimetry verification or address corresponding dosimetry uncertainties for dose response-assays. Accordingly, this study was conducted with the purpose of completing a dosimetry assessment of the radiation field within the sample chamber of a sealed source irradiator, to subsequently determine the radiation dose required to inactivate pathogenic cultures. Physical dosimetry techniques (Fricke dosimetry, ion chamber measurements, and measurements with thermoluminescent dosimeters) were used to measure dose rate and rate variances within the sample chamber. By comparing the variances between the dosimetry methodologies and measurements, an estimated dose rate within the sample chamber was determined. The results of the dosimetry evaluation were used to determine the radiation dose samples of Mycobacterium tuberculosis received, to accurately associate biological markers of inactivation to specific doses of ionizing radiation. A D10 value and dose-response curve were developed to describe the inactivation of Mtb from increasing doses of ionizing radiation. The D10 value is experimentally relevant for comparative analysis and potentially provides a biological baseline for inactivation verification. This methodology can also easily be translated to other pathogen models. ImportanceThis work set out to give us a better understanding of how much radiation is required to inactivate Mycobacterium tuberculosis, the bacteria that causes tuberculosis disease. Radiation dose from a source is not something that can just be inputted, it must be calculated, so we also determined the approximate dose from the source to address ambiguities that had previously existed while inactivating microbes. We were able to generate an accurate description of inactivation of Mycobacterium tuberculosis by correlating it with a value representing 90% death of the treated cells. We also unexpectedly discovered that very low levels of radiation increase certain activity within the cell. This is important because it allows us to better understand how radiation kills Mycobacterium tuberculosis, and gives us a value to compare to other organisms. It also offers other researchers a method to use under their own specific conditions.

microbiology↗