Stepwise differentiation of follicular helper T cells reveals distinct developmental and functional states.

Follicular helper T (Tfh) cells are essential for the formation of high affinity antibodies after vaccination or infection. Although the signals responsible for initiating Tfh differentiation from naïve T cells have been studied, the signals controlling sequential developmental stages culminating in optimal effector function are not well understood. Here we use fate mapping strategies for the cytokine IL-21 to uncover sequential developmental stages of Tfh differentiation including a progenitor-like stage, a fully developed effector stage and a post-effector Tfh stage that maintains transcriptional and epigenetic features without IL-21 production. We find that progression through these stages are controlled intrinsically by the transcription factor FoxP1 and extrinsically by follicular regulatory T cells. Through selective deletion of Tfh stages, we show that these cells control antibody dynamics during distinct stages of the germinal center reaction in response to a SARS-CoV-2 vaccine. Together, these studies demonstrate the sequential phases of Tfh development and how they promote humoral immunity.

High-resolution photocatalytic mapping of SARS-CoV-2 spike interactions on the cell surface.

Identifying virus-host interactions on the cell surface can improve our understanding of viral entry and pathogenesis. SARS-CoV-2, the causative agent of the COVID-19 disease, uses ACE2 as a receptor to enter cells. Yet the full repertoire of cell surface proteins that contribute to viral entry is unknown. We developed a photocatalyst-based viral-host protein microenvironment mapping platform (ViraMap) to probe the molecular neighborhood of the SARS-CoV-2 spike protein on the human cell surface. Application of ViraMap to ACE2-expressing cells captured ACE2, the established co-receptor NRP1, and several novel cell surface proteins. We systematically analyzed the relevance of these candidate proteins to SARS-CoV-2 entry by knockdown and overexpression approaches in pseudovirus and authentic infection models and identified PTGFRN and EFNB1 as bona fide viral entry factors. Our results highlight additional host targets that participate in SARS-CoV-2 infection and showcase ViraMap as a powerful platform for defining viral interactions on the cell surface.

Targeted proximity-labelling of protein tyrosines via flavin-dependent photoredox catalysis with mechanistic evidence for a radical-radical recombination pathway.

Flavin-based photocatalysts such as riboflavin tetraacetate (RFT) serve as a robust platform for light-mediated protein labelling via phenoxy radical-mediated tyrosine-biotin phenol coupling on live cells. To gain insight into this coupling reaction, we conducted detailed mechanistic analysis for RFT-photomediated activation of phenols for tyrosine labelling. Contrary to previously proposed mechanisms, we find that the initial covalent binding step between the tag and tyrosine is not radical addition, but rather radical-radical recombination. The proposed mechanism may also explain the mecha-nism of other reported tyrosine-tagging approaches. Competitive kinetics experiments show that phenoxyl radicals are generated with several reactive intermediates in the proposed mechanism-primarily with the excited riboflavin-photocatalyst or singlet oxygen-and these multiple pathways for phenoxyl radical generation from phenols increase the likelihood of radical-radical recombination.

Proteomic mapping of intercellular synaptic environments via flavin-dependent photoredox catalysis.

Receptor-ligand interactions play essential signaling roles within intercellular contact regions. This is particularly important within the context of the immune synapse where protein communication at the surface of physically interacting T cells and antigen-presenting cells regulate downstream immune signaling responses. To identify protein microenvironments within immunological synapses, we combined a flavin-dependent photocatalytic labeling strategy with quantitative mass spectrometry-based proteomics. Using α-PD-L1 or α-PD-1 single-domain antibody (VHH)-based photocatalyst targeting modalities, we profiled protein microenvironments within the intercellular region of an immune synapse-forming co-culture system. In addition to enrichment of both PD-L1 and PD-1 with either targeting modality, we also observed enrichment of both known immune synapse residing receptor-ligand pairs and surface proteins, as well as previously unknown synapse residing proteins.

Small molecule photocatalysis enables drug target identification via energy transfer.

Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.

Nanoscale Mapping of EGFR and c-MET Protein Environments on Lung Cancer Cell Surfaces via Therapeutic Antibody Photocatalyst Conjugates.

Receptor tyrosine kinases are involved in essential signaling roles that impact cell growth, differentiation, and proliferation. The overexpression or mutation of these proteins can lead to aberrant signaling that has been directly linked to a number of diseases including cancer cell formation and progression. This has led to intense clinical focus on modulating RTK activity through direct targeting of signaling activity or cell types harboring aberrant RTK behavior. In particular, epidermal growth factor receptor (EGFR) has attracted intense clinical attention due to the impact of inhibiting this RTK on tumor growth. However, mutations incurred through targeting EGFR have led to therapeutic resistance that involves not only direct mutations to the EGFR protein but also the involvement of other RTKs, such as c-MET, that can overcome therapeutic-based EGFR inhibition effects. This has, not surprisingly, led to co-targeting strategies of RTKs such as EGFR and c-MET to overcome resistance mechanisms. While the ability to co-target these proteins has led to success in the clinic, a more comprehensive understanding of their proximal environments, particularly in the context of therapeutic modalities, could further enhance both our understanding of their signaling biology and provide additional avenues for targeting these surface proteins. Thus, to investigate EGFR and c-MET protein microenvironments, we utilized our recently developed iridium photocatalyst-based microenvironment mapping technology to catalog EGFR and c-MET surface environments on non-small cell lung cancer cell lines. Through this approach, we enriched EGFR and c-MET from the cell surface and identified known EGFR and c-MET associators as well as previously unidentified proximal proteins.

Detection of cell-cell interactions via photocatalytic cell tagging.

The growing appreciation of immune cell-cell interactions within disease environments has led to extensive efforts to develop immunotherapies. However, characterizing complex cell-cell interfaces in high resolution remains challenging. Thus, technologies leveraging therapeutic-based modalities to profile intercellular environments offer opportunities to study cell-cell interactions with molecular-level insight. We introduce photocatalytic cell tagging (PhoTag) for interrogating cell-cell interactions using single-domain antibodies (VHHs) conjugated to photoactivatable flavin-based cofactors. Following irradiation with visible light, the flavin photocatalyst generates phenoxy radical tags for targeted labeling. Using this technology, we demonstrate selective synaptic labeling across the PD-1/PD-L1 axis in antigen-presenting cell-T cell systems. In combination with multiomics single-cell sequencing, we monitored interactions between peripheral blood mononuclear cells and Raji PD-L1 B cells, revealing differences in transient interactions with specific T cell subtypes. The utility of PhoTag in capturing cell-cell interactions will enable detailed profiling of intercellular communication across different biological systems.

Follicular T cells optimize the germinal center response to SARS-CoV-2 protein vaccination in mice.

Follicular helper T (Tfh) cells promote, whereas follicular regulatory T (Tfr) cells restrain, germinal center (GC) reactions. However, the precise roles of these cells in the complex GC reaction remain poorly understood. Here, we perturb Tfh or Tfr cells after SARS-CoV-2 spike protein vaccination in mice. We find that Tfh cells promote the frequency and somatic hypermutation (SHM) of Spike-specific GC B cells and regulate clonal diversity. Tfr cells similarly control SHM and clonal diversity in the GC but do so by limiting clonal competition. In addition, deletion of Tfh or Tfr cells during primary vaccination results in changes in SHM after vaccine boosting. Aged mice, which have altered Tfh and Tfr cells, have lower GC responses, presenting a bimodal distribution of SHM. Together, these data demonstrate that GC responses to SARS-CoV-2 spike protein vaccines require a fine balance of positive and negative follicular T cell help to optimize humoral immunity.

Strategies for monitoring cell-cell interactions.

Multicellular organisms depend on physical cell-cell interactions to control physiological processes such as tissue formation, neurotransmission and immune response. These intercellular binding events can be both highly dynamic in their duration and complex in their composition, involving the participation of many different surface and intracellular biomolecules. Untangling the intricacy of these interactions and the signaling pathways they modulate has greatly improved insight into the biological processes that ensue upon cell-cell engagement and has led to the development of protein- and cell-based therapeutics. The importance of monitoring physical cell-cell interactions has inspired the development of several emerging approaches that effectively interrogate cell-cell interfaces with molecular-level detail. Specifically, the merging of chemistry- and biology-based technologies to deconstruct the complexity of cell-cell interactions has provided new avenues for understanding cell-cell interaction biology and opened opportunities for therapeutic development.

Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes.

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton-Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE-LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE's rim domain contribute to hemolysis, namely residues 57-75 (DR1) and residues 182-196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE-DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.

Design of a Multiuse Photoreactor To Enable Visible-Light Photocatalytic Chemical Transformations and Labeling in Live Cells.

Despite the growing use of visible-light photochemistry in both chemistry and biology, no general low-heat photoreactor for use across these different disciplines exists. Herein, we describe the design and use of a standardized photoreactor for visible-light-driven activation and photocatalytic chemical transformations. Using this single benchtop photoreactor, we performed photoredox reactions across multiple visible light wavelengths, a high-throughput photocatalytic cross-coupling reaction, and in vitro labeling of proteins and live cells. Given the success of this reactor in all tested applications, we envision that this multi-use photoreactor will be widely used in biology, chemical biology, and medicinal chemistry settings.

Microenvironment mapping via Dexter energy transfer on immune cells.

Many disease pathologies can be understood through the elucidation of localized biomolecular networks, or microenvironments. To this end, enzymatic proximity labeling platforms are broadly applied for mapping the wider spatial relationships in subcellular architectures. However, technologies that can map microenvironments with higher precision have long been sought. Here, we describe a microenvironment-mapping platform that exploits photocatalytic carbene generation to selectively identify protein-protein interactions on cell membranes, an approach we term MicroMap (µMap). By using a photocatalyst-antibody conjugate to spatially localize carbene generation, we demonstrate selective labeling of antibody binding targets and their microenvironment protein neighbors. This technique identified the constituent proteins of the programmed-death ligand 1 (PD-L1) microenvironment in live lymphocytes and selectively labeled within an immunosynaptic junction.

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

Vibrio cholerae Outer Membrane Vesicles Inhibit Bacteriophage Infection.

Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (phage) treatment has been proposed as an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown. Here, we show that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent. Our results suggest that phage therapy or prophylaxis should take into consideration the production of OMVs as a bacterial decoy mechanism that could influence the outcome of phage treatment.IMPORTANCE Phages have been increasingly recognized for the significance of their interactions with bacterial cells in multiple environments. Bacteria use myriad strategies to defend against phage infection, including restriction modification, abortive infection, phase variation of cell surface receptors, phage-inducible chromosomal islands, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems. The data presented here suggest that the apparently passive process of OMV release can also contribute to phage defense. By considering the effect of OMVs on V. cholerae infection by three unique virulent phages, ICP1, ICP2, and ICP3, we show that, in vitro, a reproducible reduction in bacterial killing is both dose and phage receptor dependent. This work supports a role for OMVs as natural decoys to defend bacteria from phage predation.

Roles of Staphylococcus aureus Mnh1 and Mnh2 Antiporters in Salt Tolerance, Alkali Tolerance, and Pathogenesis.

Staphylococcus aureus has three types of cation/proton antiporters. The type 3 family includes two multisubunit Na+/H+ (Mnh) antiporters, Mnh1 and Mnh2. These antiporters are clusters of seven hydrophobic membrane-bound protein subunits. Mnh antiporters play important roles in maintaining cytoplasmic pH in prokaryotes, enabling their survival under extreme environmental stress. In this study, we investigated the physiological roles and catalytic properties of Mnh1 and Mnh2 in S. aureus Both Mnh1 and Mnh2 were cloned separately into a pGEM3Z+ vector in the antiporter-deficient KNabc Escherichia coli strain. The catalytic properties of the antiporters were measured in everted (inside out) vesicles. The Mnh1 antiporter exhibited a significant exchange of Na+/H+ cations at pH 7.5. Mnh2 showed a significant exchange of both Na+/H+ and K+/H+ cations, especially at pH 8.5. Under elevated salt conditions, deletion of the mnhA1 gene resulted in a significant reduction in the growth rate of S. aureus in the range of pH 7.5 to 9. Deletion of mnhA2 had similar effects but mainly in the range of pH 8.5 to 9.5. Double deletion of mnhA1 and mnhA2 led to a severe reduction in the S. aureus growth rate mainly at pH values above 8.5. The effects of functional losses of both antiporters in S. aureus were also assessed via their support of virulence in a mouse in vivo infection model. Deletion of the mnhA1 gene led to a major loss of S. aureus virulence in mice, while deletion of mnh2 led to no change in virulence.IMPORTANCE This study focuses on the catalytic properties and physiological roles of Mnh1 and Mnh2 cation/proton antiporters in S. aureus and their contributions under different stress conditions. The Mnh1 antiporter was found to have catalytic activity for Na+/H+ antiport, and it plays a significant role in maintaining halotolerance at pH 7.5 while the Mnh2 antiporter has catalytic antiporter activities for Na+/H+ and K+/H+ that have roles in both osmotolerance and halotolerance in S. aureus Study of S. aureus with a single deletion of either mnhA1 or mnhA2 was assessed in an infection model of mice. The result shows that mnhA1, but not mnhA2, plays a major role in S. aureus virulence.

Staphylococcus aureus Pore-Forming Toxins.

Staphylococcus aureus (S. aureus) is a formidable foe equipped with an armamentarium of virulence factors to thwart host defenses and establish a successful infection. Among these virulence factors, S. aureus produces several potent secreted proteins that act as cytotoxins, predominant among them the beta-barrel pore-forming toxins. These toxins play several roles in pathogenesis, including disruption of cellular adherens junctions at epithelial barriers, alteration of intracellular signaling events, modulation of host immune responses, and killing of eukaryotic immune and non-immune cells. This chapter provides an updated overview on the S. aureus beta-barrel pore-forming cytotoxins, the identification of toxin receptors on host cells, and their roles in pathogenesis.

Staphylococcus aureus Leukocidin LukED and HIV-1 gp120 Target Different Sequence Determinants on CCR5.

Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5. IMPORTANCE: The bicomponent pore-forming toxins are thought to play a vital role in the success of Staphylococcus aureus as a mammalian pathogen. One of the leukocidins, LukED, is necessary and sufficient for lethality in mice. At the molecular level, LukED causes cell lysis through binding to specific cellular receptors. CCR5 is one of the receptors targeted by LukED and is the major coreceptor for CCR5-tropic HIV-1. While the molecular interaction of CCR5 and HIV-1 is well characterized, the means by which LukED interacts with CCR5 is less clear. In this study, we demonstrated that receptor specificity is conferred through unique interactions between key domains on CCR5 and LukE. Although HIV-1 and LukED target the same receptor, our data demonstrated that they interact with CCR5 differently, highlighting the molecular complexity of host-pathogen interactions.

Staphylococcus aureus Coordinates Leukocidin Expression and Pathogenesis by Sensing Metabolic Fluxes via RpiRc.

UNLABELLED: Staphylococcus aureus is a formidable human pathogen that uses secreted cytolytic factors to injure immune cells and promote infection of its host. Of these proteins, the bicomponent family of pore-forming leukocidins play critical roles in S. aureus pathogenesis. The regulatory mechanisms governing the expression of these toxins are incompletely defined. In this work, we performed a screen to identify transcriptional regulators involved in leukocidin expression in S. aureus strain USA300. We discovered that a metabolic sensor-regulator, RpiRc, is a potent and selective repressor of two leukocidins, LukED and LukSF-PV. Whole-genome transcriptomics, S. aureus exoprotein proteomics, and metabolomic analyses revealed that RpiRc influences the expression and production of disparate virulence factors. Additionally, RpiRc altered metabolic fluxes in the trichloroacetic acid cycle, glycolysis, and amino acid metabolism. Using mutational analyses, we confirmed and extended the observation that RpiRc signals through the accessory gene regulatory (Agr) quorum-sensing system in USA300. Specifically, RpiRc represses the rnaIII promoter, resulting in increased repressor of toxins (Rot) levels, which in turn negatively affect leukocidin expression. Inactivation of rpiRc phenocopied rot deletion and increased S. aureus killing of primary human polymorphonuclear leukocytes and the pathogenesis of bloodstream infection in vivo. Collectively, our results suggest that S. aureus senses metabolic shifts by RpiRc to differentially regulate the expression of leukocidins and to promote invasive disease. IMPORTANCE: The bicomponent pore-forming leukocidins play pivotal roles in the ability of S. aureus to kill multiple host immune cells, thus enabling this pathogen to have diverse tissue- and species-tropic effects. While the mechanisms of leukocidin-host receptor interactions have been studied in detail, the regulatory aspects of leukocidin expression are less well characterized. Moreover, the expression of the leukocidins is highly modular in vitro, suggesting the presence of regulators other than the known Agr, Rot, and S. aureus exoprotein pathways. Here, we describe how RpiRc, a metabolite-sensing transcription factor, mediates the repression of two specific leukocidin genes, lukED and pvl, which in turn has complex effects on the pathogenesis of S. aureus Our findings highlight the intricacies of leukocidin regulation by S. aureus and demonstrate the involvement of factors beyond traditional virulence factor regulators.

Exploiting dominant-negative toxins to combat Staphylococcus aureus pathogenesis.

Staphylococcus aureus (S. aureus) is a human pathogen that relies on the subversion of host phagocytes to support its pathogenic lifestyle. S. aureus strains can produce up to five beta-barrel, bi-component, pore-forming leukocidins that target and kill host phagocytes. Thus, preventing immune cell killing by these toxins is likely to boost host immunity. Here, we describe the identification of glycine-rich motifs within the membrane-penetrating stem domains of the leukocidin subunits that are critical for killing primary human neutrophils. Remarkably, leukocidins lacking these glycine-rich motifs exhibit dominant-negative inhibitory effects toward their wild-type toxin counterparts as well as other leukocidins. Biochemical and cellular assays revealed that these dominant-negative toxins work by forming mixed complexes that are impaired in pore formation. The dominant-negative leukocidins inhibited S. aureus cytotoxicity toward primary human neutrophils, protected mice from lethal challenge by wild-type leukocidin, and reduced bacterial burden in a murine model of bloodstream infection. Thus, we describe the first example of staphylococcal bi-component dominant-negative toxins and their potential as novel therapeutics to combat S. aureus infection.