JoeyKelly

bioRxiv [Preprint]. 2025 Sep 27:2025.09.26.678899. doi: 10.1101/2025.09.26.678899.

ABSTRACT

Bordetella pertussis, the etiologic agent of whooping cough, remains a serious public health concern despite widespread vaccination. Improved therapeutics and vaccines are urgently needed to treat and prevent pertussis disease. Host recognition of bacterial peptidoglycan (PGN), including B. pertussis extracellular PGN fragment tracheal cytotoxin (TCT), shapes the immune response to infection. Peptidoglycan recognition proteins (PGLYRPs) are a conserved family of innate immune molecules which bind bacterial PGN. While they function as immune signaling receptors in arthropods, PGLYRPs in mammals have thus far been primarily recognized for their bactericidal activity. Previously thought to function only as antimicrobial peptides in mammals, the immune modulatory roles of this family of peptidoglycan recognition proteins are beginning to gain greater appreciation. Peptidoglycan recognition protein 1 (PGLYRP1) is a secreted antimicrobial protein. However, its role in mammalian host defenses and immune signaling during infection with Gram-negative pathogens, such as B. pertussis, remain largely unknown. Here, we identify a dual role for PGLYRP1 in modulating host immune responses to B. pertussis. Using knockout mice, single-cell and bulk transcriptomics and functional assays, we show that PGLYRP1 has bactericidal activity against B. pertussis in vitro and promotes early bacterial control in vivo. PGLYRP1 also dampens inflammatory responses and impedes bacterial killing later in infection. Mechanistically, PGLYRP1 enhances nucleotide oligomerization domain (NOD)-1 signaling in response to TCT while suppressing NOD2- and triggering receptor expressed on myeloid cells-1 (TREM-1)-mediated inflammatory pathways. TCT-bound PGLYRP1 selectively impairs TREM-1 activation compared to PGNs from other bacteria, revealing a novel bacterial immune evasion strategy. These findings demonstrate that B. pertussis co-opts PGLYRP1 to temper inflammation and alter immune signaling, revealing a novel immune evasion mechanism of manipulating the availability and structure of their exogenous peptidoglycan, revealing implications for host-pathogen evolution, vaccine design and host-directed therapeutics.

PMID:41040336 | PMC:PMC12485853 | DOI:10.1101/2025.09.26.678899

bioRxiv [Preprint]. 2024 Oct 3:2024.10.02.616386. doi: 10.1101/2024.10.02.616386.

ABSTRACT

No currently licensed vaccine reliably prevents pulmonary tuberculosis (TB), a leading cause of infectious disease mortality. Developing effective new vaccines will require identifying which of the roughly 4000 proteins in the Mycobacterium tuberculosis ( Mtb ) proteome are presented on MHC class II (MHC-II) by infected human phagocytes and can be recognized by CD4+ T cells to mediate protective immunity. Vaccines must also elicit T cell responses recognizing the same peptide-MHC complexes presented by infected cells, and successful presentation of target human MHC-II peptides is currently challenging to evaluate and optimize. Here, we define antigenic targets for TB vaccine development by using mass spectrometry (MS) for proteome-wide discovery of Mtb epitopes presented on MHC-II by infected human cells. We next iteratively design and evaluate candidate mRNA vaccine immunogens, revealing design principles that enhance presentation of target MHC-II peptides. Our results will inform the development of new TB vaccine candidates.

PMID:41031036 | PMC:PMC12478359 | DOI:10.1101/2024.10.02.616386

ACS Omega. 2025 Aug 22;10(34):39060-39072. doi: 10.1021/acsomega.5c05358. eCollection 2025 Sep 2.

ABSTRACT

Novel immunopotentiators are essential for advancing our understanding of immune receptor crosstalk and for addressing infectious diseases. Previous studies have suggested that coactivation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 4 (TLR4) can synergistically enhance the immune response. To investigate this synergy, we synthesized and evaluated a series of conjugated NOD2/TLR4 dual agonists comprising our in-house NOD2 agonist and two structurally distinct TLR4 agonists connected via flexible or rigid linkers. Our findings indicate that dual agonist activity toward both NOD2 and TLR4 is diminished upon conjugation. We also show that the linker chemistry significantly influences the kinetic solubility of these conjugates. Furthermore, the conjugates elicit distinct immunomodulatory effects in human primary peripheral blood mononuclear cells, characterized by a Th2-polarized cytokine response. These results provide insights into the structure-activity relationship of conjugated NOD2/TLR4 agonists and offer preliminary guidelines for tuning their solubility profiles.

PMID:40918346 | PMC:PMC12409580 | DOI:10.1021/acsomega.5c05358

Cytometry A. 2025 Sep 18. doi: 10.1002/cyto.a.24959. Online ahead of print.

ABSTRACT

Cytotoxic CD8+ T cells eliminate virus-infected or cancer cells, thus playing a pivotal role in anti-viral and anti-cancer immunity. Tetramer reagents, which consist of fluorochrome-labeled streptavidin coupled with peptide-loaded MHC I molecules, enable the detection of antigen-specific CD8+ T cells using flow cytometry. The development of tetramer reagents has been instrumental for our understanding of antigen-specific CD8+ T cells and their roles in immune responses. More recently, combinatorial tetramer staining protocols have enabled the simultaneous detection and monitoring of multiple specificities and concomitant pathogen-dependent CD8+ T cell dynamics. However, these methods are either based on mass cytometry, preventing the isolation of antigen-specific CD8+ T cells for downstream investigation, or have provided a less comprehensive picture of the phenotypic characteristics of antigen-specific CD8+ T cells when based on flow cytometry. Here we describe the development of a combinatorial tetramer staining protocol in combination with high-dimensional CD8+ T cell immunophenotyping in the context of virus-specific CD8+ T cells leveraging spectral flow cytometry. Our assay enables the simultaneous measurement of 15 different CD8+ T cell specificities and includes an additional 18 markers to define the phenotypic and functional characteristics of antigen-specific CD8+ T cells. We describe our assay optimization strategies, with the goal of improving marker and tetramer resolution while eliminating sources of background noise. Finally, we apply this method to reveal the phenotypic heterogeneity of virus-specific CD8+ T cells against common viral pathogens in healthy individuals.

PMID:40964879 | DOI:10.1002/cyto.a.24959

mSphere. 2025 Sep 30;10(9):e0043125. doi: 10.1128/msphere.00431-25. Epub 2025 Sep 8.

ABSTRACT

Bacterial persisters are a subpopulation of cells that exhibit a transient non-susceptible phenotype in the presence of bactericidal antibiotic concentrations. This phenotype can lead to the survival and regrowth of bacteria after treatment, resulting in relapse of infections. It is also a contributing factor to antibacterial resistance. Multiple processes are believed to cause persister formation; however, identifying the proteins expressed during the induction of persistence is challenging because the persister state is rare, transient, and does not result in genetic changes. In this study, we used Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) to label and retrieve the proteome expressed during persistence and recovery for two strains of Staphylococcus aureus exposed to β-lactam and fluoroquinolone antibiotics. After incubating antibiotic-exposed bacteria with the methionine ortholog L-azidohomoalanine to label the proteins of persister cells, we retrieved labeled proteins using click chemistry-pulldown methodology. Analysis of the retrieved proteome of persisters with Label-Free Quantification-Liquid chromatography mass spectrometry (LFQ-LCMS)-based proteomics revealed widespread changes in translation. Our analysis uncovered previously identified persister genes, including, for example, relA/spot-system, changes in purine and amino acid metabolism, the upregulation and downregulation of transcription factors, and changes to influx and efflux pumps, thus validating our methodology. In addition, we also identified numerous novel persister-associated proteins. Few changes were conserved across the two strains and both antibiotics. Instead, results suggest that the mechanisms of persister formation vary across genotypes and the drugs to which strains are exposed. These findings provide evidence that the entry into persistence is an active process that dramatically alters the translational behavior of cells and suggest that downregulation of metabolism, by diverse but functionally similar processes, in persister cells enables cells to survive antibiotic pressure.IMPORTANCEIn this study, we have applied a technique called "Bioorthogonal Non-Canonical Amino Acid-Tagging," or BONCAT, to identify which proteins are expressed when bacteria are in the persister state. Our work makes novel contributions to our understanding of persister cells, a bacterial sub-population that gives rise to recurrent infections, and establishes BONCAT as a valuable tool to study phenotypic heterogeneity in bacterial populations.

PMID:40920103 | PMC:PMC12482189 | DOI:10.1128/msphere.00431-25

Front Immunol. 2025 Aug 12;16:1608769. doi: 10.3389/fimmu.2025.1608769. eCollection 2025.

ABSTRACT

Previously we have shown that H2-congenic recombinant mice of the B6.I-9.3 (H2-Ab1 ^j) strain are significantly more susceptible to tuberculosis (TB) infection compared to their C57BL/6 (B6, H2-A ^b) ancestors. Impaired TB control was characterized by decreased selection and maintenance of CD4⁺ T-cells, their profoundly narrower TCR repertoires, and a disproportionally enlarged neutrophil population. All phenotypes were expressed before TB infection, thus reflecting the steady state of the immune system and providing the basis of true genetic TB susceptibility. We anticipated that the differences in parameters of pre-infection immune homeostasis would seriously influence development of specific immune responses shortly after mycobacterial invasion and affect TB defense thereafter. In this study, we report on the dynamic phenotypes of CD4⁺ T-cells responding to infection which differ profoundly between mice bearing different MHC-II alleles. First, during post-challenge week 3, despite identical lung mycobacterial load, mice carrying the "resistant" H2-A ^b allele recruited significantly more mycobacteria-specific, IFN-γ-producing CD4⁺ T-cells to their lungs compared to H2-Ab1 ^j allele carriers. Second, during a few months post challenge, B6 mice were able to control both the size of the IFN-γ-producing CD4⁺ T-cell population and the total proportion of activated CD4⁺ T-cells at levels significantly lower than those in B6.I-9.3 mice. Finally, in TB-susceptible mice, a higher proportion of CD4⁺ T-cells expressed both activation-associated and immune inhibition (checkpoint) markers, accompanied by functional CD4⁺ T-cell exhaustion at late stages of infection. Together, these observations suggest that suboptimal pre-infection MHC-II-dependent shifts in immune homeostasis affect both early and late immune reactions against TB.

PMID:40873577 | PMC:PMC12378094 | DOI:10.3389/fimmu.2025.1608769

Cells. 2025 Aug 9;14(16):1231. doi: 10.3390/cells14161231.

ABSTRACT

Major histocompatibility complex class I (MHC-I)-mediated antigen presentation plays a pivotal role in anti-tumor immunity by enabling CD8⁺ T cells to recognize and eliminate malignant cells. In melanoma, modulation of this pathway is critical for improving the efficacy of immunotherapies. Our study demonstrates that the natural compound Cepharanthine (CEP) exhibits notable antitumor activity by enhancing MHC-I-mediated antigen presentation. CEP treatment upregulated MHC-I expression (both membrane-bound and total levels) in melanoma cells in a concentration-dependent manner, thereby improving antigen-presenting capacity. Interestingly, when autophagy was pharmacologically blocked using Bafilomycin A1, co-treatment with CEP did not lead to further elevation of MHC-I expression, suggesting that CEP's effect is mediated through disruption of the autophagic pathway. Mechanistically, CEP induced autophagosome accumulation, as evidenced by an increase in GFP-LC3 puncta. Fluorescence imaging further confirmed that CEP selectively impaired lysosomal acidification without affecting autophagosome-lysosome fusion, thereby inhibiting late-stage autophagic flux. Furthermore, CEP treatment promoted CD8⁺ T cell infiltration into tumor tissues and enhanced the antitumor efficacy of anti-PD-1 therapy, resulting in greater tumor suppression compared to either treatment alone. The study elucidates how CEP's selective lysosomal inhibition creates a tumor microenvironment more susceptible to immune surveillance, primarily through preserved MHC-I surface expression and subsequent T cell recognition. This work highlights CEP as a promising immunomodulatory agent and provides a potential strategy for improving the outcomes of immune checkpoint blockade therapy.

PMID:40862710 | PMC:PMC12384187 | DOI:10.3390/cells14161231