Ligation of Dectin-2 with a novel microbial ligand promotes adjuvant activity for vaccination

Wang, Huafeng; Lee, Taekjin; Fites, Scott; Merkhofer, Richard M.; Żarnowski, Robert; Brandhorst, T. Tristan; Galles, Kevin; Klein, Bruce S.; Wüthrich, Marcel

doi:10.1371/journal.ppat.1006568

Cited by 34 publications

(25 citation statements)

References 40 publications

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“…The mutation frequency of PRA1 produced by CRISPR/Cas9 editing was much higher than the approximately 0.05 to 2% typical of traditional homologous-recombination-based gene editing in Blastomyces (for example, see reference 7 ). We sought to determine whether this high targeting efficiency was locus specific, so we designed sgRNA vectors to target several other Blastomyces genes, including (i) the high-affinity zinc transporter ZRT1 , (ii) the recently identified dectin-2 ligand Blastomyces endoglucanase 2 ( BL-ENG2 ) ( 10 ), and (iii) an exogenously introduced mCherry gene in an engineered Blastomyces strain ( 11 ). For these attempts, we chose multiple sgRNA sequences for each target.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis

Kujoth

Sullivan

Merkhofer

et al. 2018

mBio

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Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo.

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Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis

Kujoth

Sullivan

Merkhofer

et al. 2018

mBio

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“…Mannose receptor recognizes mannose and other sugar moieties on the surface of microbial cells . The CLR dectin‐2 has previously been shown to respond to mannan stimulation, and recent work has also identified the glycoprotein Bl‐Eng2 of Blasotmyces dermatitidis as a bona‐fide ligand for this receptor . A newly defined CLR, MelLec enables the host to sense melanin on pathogens including Aspergillus spores .…”

Section: Fungal Recognition and T‐cell Priming The Lungmentioning

confidence: 99%

Helper T‐cell responses and pulmonary fungal infections

McDermott

Klein

2018

Immunology

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The mucosal surface of the respiratory tract encounters microbes, such as fungal particles, with every inhaled breath. When pathogenic fungi breach the physical barrier and innate immune system within the lung to establish an infection, adaptive immunity is engaged, often in the form of helper CD4 T-cell responses. Type 1 responses, characterized by interferon-γ production from CD4 cells, promote clearance of Histoplasma capsulatum and Cryptococcus neoformans infection. Likewise, interleukin-17A (IL-17A) production from Th17 cells promotes immunity to Blastomyces dermatitidis and Coccidioides species infection by recruiting neutrophils. In contrast the development of T helper type 2 responses, characterized by IL-5 production from T cells and eosinophil influx into the lungs, drives allergic bronchopulmonary aspergillosis and poor outcomes during C. neoformans infection. Experimental vaccines against several endemic mycoses, including Histoplasma capsulatum, Coccidioides, Cryptococcus and Blastomyces dermatitidis, induce protective T-cell responses and foreshadow the development of vaccines against pulmonary fungal infections for use in humans. Additionally, recent work using antifungal T cells as immunotherapy to protect immune-compromised patients from opportunist fungal infections also shows great promise. This review covers the role of T-cell responses in driving protection and pathology in response to pulmonary fungal infections, and highlights promising therapeutic applications of antifungal T cells.

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“…For instance, Mincle recognizes SAP30, a protein released by dead cells (33). Moreover, we observed that digestion of the crude, water-soluble, cell wall extract (CWE) from Blastomyces vaccine yeast with either proteinase K or endo-mannosidases reduced Dectin-2 recognition as measured by reduced ligand activity by corresponding reporter cells (23), suggesting that both the protein and glycan moieties may contribute.…”

Section: Dectin-2 Recognizes Mannose Residues In Bl-eng2 But Not Thementioning

confidence: 96%

“…4C-F). As a positive control for CD4 + T cell priming, we vaccinated mice with full length Bl-Eng2 since it harbors both the antigen and adjuvant properties (23,34).…”

Section: Dectin-2 Senses Bl-eng2's C-terminal O-linked Glycans But Nmentioning

confidence: 99%

Structural basis ofBlastomycesEndoglucanase-2 adjuvancy in anti-fungal and -viral immunity

Dias

Dobson

Bakke

et al. 2020

Preprint

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The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4 + and CD8 + T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit induced protective immunity against lethal influenza and fungal pulmonary challenge. AUTHOR SUMMARYFungal disease remains a challenging clinical and public health problem in part because there is no commercial vaccine available. The lack of suitable adjuvants is a critical barrier to developing safe and effective vaccines against fungal pathogens. Current adjuvants such as alum preferentially induce antibody responses which may be limited in mediating protection against fungi. Clinical observations and animal studies implicate cellular immunity as the essential component for the resolution of fungal infections. We have recently discovered an adjuvant that augments cell mediated immune responses and vaccine induced protection against fungi. Here, we identified the structural and mechanistic requirements by which this newly discovered adjuvant induces cell mediated immunity against fungi. As a proof of principle we also demonstrate that the adjuvant drives cellular immune responses against viruses such as influenza. We anticipate that our adjuvant can be used for vaccination with safe subunit vaccines against many microbial pathogens including viruses, intracellular bacteria, fungi and parasites that require cell mediated immune responses.We recently demonstrated that fungal recognition by C-type lectin receptor (CLR) Dectin-2 is required for the differentiation of protective, antifungal Th1 and Th17 cells (16). Dectin-2 is expressed by macrophages, dendritic cells (DCs), neutrophils, monocytes, and other myeloid cells that binds high-mannose structures displayed on the surface of microorganisms, particularly fungi (17,18). The extracellular carbohydrate-recognition domain (CRD) of Dectin-2 shows high affinity for binding α-1,2 and α-1,4 mannose in a Ca 2+ -dependent fashion (19) and, together with FcRγ, triggers intracellular signaling (20). Few Dectin-2 ligands have been described, including MP98, a protein of Cryptococcus that is highly modifi...

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Ligation of Dectin-2 with a novel microbial ligand promotes adjuvant activity for vaccination

Cited by 34 publications

References 40 publications

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis

Helper T‐cell responses and pulmonary fungal infections

Structural basis ofBlastomycesEndoglucanase-2 adjuvancy in anti-fungal and -viral immunity

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