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.
Leukocidins and the Nuclease Nuc Prevent Neutrophil-Mediated Killing of Staphylococcus aureus Biofilms
Bacterial biofilms are linked with chronic infections and have properties distinct from those of planktonic, single-celled bacteria. The virulence mechanisms associated with Staphylococcus aureus biofilms are becoming better understood. Human neutrophils are critical for the innate immune response to S. aureus infection. Here, we describe two virulence strategies that converge to promote the ability of S. aureus biofilms to evade killing by neutrophils. Specifically, we show that while neutrophils exposed to S. aureus biofilms produce extracellular traps (NETs) and phagocytose bacteria, both mechanisms are inefficient in clearance of the biofilm biomass. This is attributed to the leukocidin LukAB, which promotes S. aureus survival during phagocytosis. We also show that the persistence of biofilm bacteria trapped in NETs is facilitated by S. aureus nuclease (Nuc)-mediated degradation of NET DNA. This study describes key aspects of the interaction between primary human neutrophils and S. aureus biofilms and provides insight into how S. aureus evades the neutrophil response to cause persistent infections.
Targeting leukocidin-mediated immune evasion protects mice from Staphylococcus aureus bacteremia
Staphylococcus aureus is responsible for various diseases in humans, and recurrent infections are commonly observed. S. aureus produces an array of bicomponent pore-forming toxins that target and kill leukocytes, known collectively as the leukocidins. The contribution of these leukocidins to impair the development of anti-S. aureus adaptive immunity and facilitate reinfection is unclear. Using a murine model of recurrent bacteremia, we demonstrate that infection with a leukocidin mutant results in increased levels of anti-S. aureus antibodies compared with mice infected with the WT parental strain, indicating that leukocidins negatively impact the generation of anti-S. aureus antibodies in vivo. We hypothesized that neutralizing leukocidin-mediated immune subversion by vaccination may shift this host-pathogen interaction in favor of the host. Leukocidin-immunized mice produce potent leukocidin-neutralizing antibodies and robust Th1 and Th17 responses, which collectively protect against bloodstream infections. Altogether, these results demonstrate that blocking leukocidin-mediated immune evasion can promote host protection against S. aureus bloodstream infection.
An Intestinal Organoid-Based Platform That Recreates Susceptibility to T Cell-Mediated Tissue Injury
A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that co-cultured allogeneic T cells kill Atg16L1 mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.
Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus Î³-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope
Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the Î³-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved Î²-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.
Distinct Features of Human Myeloid Cell Cytokine Response Profiles Identify Neutrophil Activation by Cytokines as a Prognostic Feature during Tuberculosis and Cancer
Myeloid cells are a vital component of innate immunity and comprise monocytes, macrophages, dendritic cells, and granulocytes. How myeloid cell lineage affects activation states in response to cytokines remains poorly understood. The cytokine environment and cellular infiltrate during an inflammatory response may contain prognostic features that predict disease outcome. In this study, we analyzed the transcriptional responses of human monocytes, macrophages, dendritic cells, and neutrophils in response to stimulation by IFN-Î³, IFN-Î², IFN-Î», IL-4, IL-13, and IL-10 cytokines to better understand the heterogeneity of activation states in inflammatory conditions. This generated a myeloid cell-cytokine-specific response matrix that can infer representation of myeloid cells and the cytokine environment they encounter during infection, in tumors and in whole blood. Neutrophils were highly responsive to type 1 and type 2 cytokine stimulation but did not respond to IL-10. We identified transcripts specific to IFN-Î² stimulation, whereas other IFN signature genes were upregulated by both IFN-Î³ and IFN-Î². When we used our matrix to deconvolute blood profiles from tuberculosis patients, the IFN-Î²-specific neutrophil signature was reduced in tuberculosis patients with active disease, whereas the shared response to IFN-Î³ and IFN-Î² in neutrophils was increased. When applied to glioma patients, transcripts of neutrophils exposed to IL-4/IL-13 and monocyte responses to IFN-Î³ or IFN-Î² emerged as opposing predictors of patient survival. Hence, by dissecting how different myeloid cells respond to cytokine activation, we can delineate biological roles for myeloid cells in different cytokine environments during disease processes, especially during infection and tumor progression.
Structure-based discovery of a small-molecule inhibitor of methicillin-resistant Staphylococcus aureus virulence
TheÂ rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton-Valentine leukocidin (PVL) and Î±-toxin are exotoxins that create lytic pores in the host cell membrane.Â They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and Î±-toxin in their soluble, monomeric and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC).Â The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and Î±-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.
All major cholesterol-dependent cytolysins use glycans as cellular receptors
Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P1 antigen and Î±Gal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF O-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.
Unbiased identification of immunogenic Staphylococcus aureus leukotoxin B-cell epitopes
Unbiased identification of individual, immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as little as a single major pathogen antigen. Using the bi-component pore-forming leukocidin (Luks) exotoxins of the major pathogen Staphylococcus aureus (Sa) as a prototype, we randomly fragmented and separately ligated the Hemolysin gamma A (HlgA) and LukS genes into a custom-built, phage-display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk mAb recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino acid sequence that is highly conserved in four Luk toxin subunits, and is ubiquitous in representation within Sa clinical isolates. The isolated 11-amino acid peptide probe was predicted to retain the native 3D-conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk mAb, and using mutated peptides we showed that a particular amino acid side-chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG-responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment, phage-display based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much needed Sa-protective subunit vaccine.
Repurposed Drugs That Block the Gonococcus-Complement Receptor 3 Interaction Can Prevent and Cure Gonococcal Infection of Primary Human Cervical Epithelial Cells
In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.IMPORTANCE Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal-I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections.