Vaccine protection by Cryptococcus neoformans Δsgl1 is mediated by γδ T cells via TLR2 signaling.

We previously reported that administration of Cryptococcus neoformans Δsgl1 mutant vaccine, accumulating sterylglucosides (SGs) and having normal capsule (GXM), protects mice from a subsequent infection even during CD4+ T cells deficiency, a condition commonly associated with cryptococcosis. Here, we studied the immune mechanism that confers host protection during CD4+T deficiency. Mice receiving Δsgl1 vaccine produce IFNγ and IL-17A during CD4+ T (or CD8+ T) deficiency, and protection was lost when either cytokine was neutralized. IFNγ and/or IL-17A are produced by γδ T cells, and mice lacking these cells are no longer protected. Interestingly, ex vivo γδ T cells are highly stimulated in producing IFNγ and/or IL-17A by Δsgl1 vaccine, but this production was significantly decreased when cells were incubated with C. neoformans Δcap59/Δsgl1 mutant, accumulating SGs but lacking GXM. GXM modulates toll-like receptors (TLRs), including TLR2. Importantly, neither Δsgl1 nor Δcap59/Δsgl1 stimulate IFNγ or IL-17A production by ex vivo γδ T cells from TLR2-/- mice. Finally, TLR2-/- animals do not produce IL-17A in response to Δsgl1 vaccine and were no longer protected from WT challenge. Our results suggest that SGs may act as adjuvants for GXM to stimulate γδ T cells in producing IFNγ and IL-17A via TLR2, a mechanism that is still preserved upon CD4+ T deficiency.

Vaccination with Live or Heat-Killed Aspergillus fumigatus ΔsglA Conidia Fully Protects Immunocompromised Mice from Invasive Aspergillosis.

Aspergillus fumigatus causes invasive aspergillosis (IA) in immunocompromised patients, resulting in high mortality rates. Currently, no vaccine formulations to promote immune protection in at-risk individuals have been developed. In this work, we deleted the sterylglucosidase-encoding gene, sglA, in Aspergillus fumigatus and investigated its role in fungal virulence and host vaccine protection. The ΔsglA mutant accumulated sterylglucosides (SGs), newly studied immunomodulatory glycolipids, and exhibited reduced hyphal growth and altered compositions of cell wall polysaccharides. Interestingly, the ΔsglA mutant was avirulent in two murine models of IA and was fully eliminated from the lungs. Both corticosteroid-induced immunosuppressed and cyclophosphamide-induced leukopenic mice vaccinated with live or heat-killed ΔsglA conidia were fully protected against a lethal wild-type A. fumigatus challenge. These results highlight the potential of SG-accumulating strains as safe and promising vaccine formulations against invasive fungal infections. IMPORTANCE Infections by Aspergillus fumigatus occur by the inhalation of environmental fungal spores called conidia. We found that live mutant conidia accumulating glycolipids named sterylglucosides are not able to cause disease when injected into the lung. Interestingly, these animals are now protected against a secondary challenge with live wild-type conidia. Remarkably, protection against a secondary challenge persists even with vaccination with heat-killed mutant conidia. These results will significantly advance the field of the research and development of a safe fungal vaccine for protection against the environmental fungus A. fumigatus.

Comparative genomics of the plant-growth promoting bacterium Sphingobium sp. strain AEW4 isolated from the rhizosphere of the beachgrass Ammophila breviligulata.

BACKGROUND: The genus Sphingobium within the class Alpha-proteobacteria contains a small number of plant-growth promoting rhizobacteria (PGPR), although it is mostly comprised of organisms that play an important role in biodegradation and bioremediation in sediments and sandy soils. A Sphingobium sp. isolate was obtained from the rhizosphere of the beachgrass Ammophila breviligulata with a variety of plant growth-promoting properties and designated as Sphingobium sp. strain AEW4. RESULTS: Analysis of the 16S rRNA gene as well as full genome nucleotide and amino acid identities revealed that this isolate is most similar to Sphingobium xenophagum and Sphingobium hydrophobicum. Comparative genomics analyses indicate that the genome of strain AEW4 contains unique features that explain its relationship with a plant host as a PGPR, including pathways involved in monosaccharide utilization, fermentation pathways, iron sequestration, and resistance to osmotic stress. Many of these unique features are not broadly distributed across the genus. In addition, pathways involved in the metabolism of salicylate and catechol, phenyl acetate degradation, and DNA repair were also identified in this organism but not in most closely related organisms. CONCLUSION: The genome of Sphingobium sp. strain AEW4 contains a number of distinctive features that are crucial to explain its role as a plant-growth promoting rhizobacterium, and comparative genomics analyses support its classification as a relevant Sphingobium strain involved in plant growth promotion of beachgrass and other plants.

Three Models of Vaccination Strategies Against Cryptococcosis in Immunocompromised Hosts Using Heat-Killed Cryptococcus neoformans Δsgl1.

Vaccines are one of the greatest medical accomplishments to date, yet no fungal vaccines are currently available in humans mainly because opportunistic mycoses generally occur during immunodeficiencies necessary for vaccine protection. In previous studies, a live, attenuated Cryptococcus neoformans Δsgl1 mutant accumulating sterylglucosides was found to be avirulent and protected mice from a subsequent lethal infection even in absence of CD4+ T cells, a condition most associated with cryptococcosis (e.g., HIV). Here, we tested three strategies of vaccination against cryptococcosis. First, in our preventative model, protection was achieved even after a 3-fold increase of the vaccination window. Second, because live C. neoformans Δsgl1-vaccinated mice challenged more than once with WT strain had a significant decrease in lung fungal burden, we tested C. neoformans Δsgl1 as an immunotherapeutic. We found that therapeutic administrations of HK C. neoformans Δsgl1 post WT challenge significantly improves the lung fungal burden. Similarly, therapeutic administration of HK C. neoformans Δsgl1 post WT challenge resulted in 100% or 70% survival depending on the time of vaccine administration, suggesting that HK Δsgl1 is a robust immunotherapeutic option. Third, we investigated a novel model of vaccination in preventing reactivation from lung granuloma using C. neoformans Δgcs1. Remarkably, we show that administration of HK Δsgl1 prevents mice from reactivating Δgcs1 upon inducing immunosuppression with corticosteroids or by depleting CD4+ T cells. Our results suggest that HK Δsgl1 represents a clinically relevant, efficacious vaccine that confers robust host protection in three models of vaccination against cryptococcosis even during CD4-deficiency.

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

Cryptococcus neoformans Δsgl1 Vaccination Requires Either CD4+ or CD8+ T Cells for Complete Host Protection.

Cryptococcus neoformans is a fungal pathogen causing life-threatening meningoencephalitis in susceptible individuals. Fungal vaccine development has been hampered by the fact that cryptococcosis occurs during immunodeficiency. We previously reported that a C. neoformans mutant (Δsgl1) accumulating sterylglucosides (SGs) is avirulent and provides complete protection to WT challenge, even under CD4+ T cell depletion, an immunodeficient condition commonly associated with cryptococcosis. We found high levels of SGs in the lungs post-immunization with Δsgl1 that decreased upon fungal clearance. Th1 cytokines increased whereas Th2 cytokines concurrently decreased, coinciding with a large recruitment of leukocytes to the lungs. Depletion of B or CD8+ T cells did not affect either Δsgl1 clearance or protection from WT challenge. Although CD4+ T cell depletion affected clearance, mice were still protected indicating that clearance of the mutant was not necessary for host protection. Protection was lost only when both CD4+ and CD8+ T cells were depleted, highlighting a previously unexplored role of fungal-derived SGs as an immunoadjuvant for host protection against cryptococcosis.

Animal Models of Cryptococcus neoformans in Identifying Immune Parameters Associated With Primary Infection and Reactivation of Latent Infection.

Cryptococcus species are environmental fungal pathogens and the causative agents of cryptococcosis. Infection occurs upon inhalation of infectious particles, which proliferate in the lung causing a primary infection. From this primary lung infection, fungal cells can eventually disseminate to other organs, particularly the brain, causing lethal meningoencephalitis. However, in most cases, the primary infection resolves with the formation of a lung granuloma. Upon severe immunodeficiency, dormant cryptococcal cells will start proliferating in the lung granuloma and eventually will disseminate to the brain. Many investigators have sought to study the protective host immune response to this pathogen in search of host parameters that keep the proliferation of cryptococcal cells under control. The majority of the work assimilates research carried out using the primary infection animal model, mainly because a reactivation model has been available only very recently. This review will focus on anti-cryptococcal immunity in both the primary and reactivation models. An understanding of the differences in host immunity between the primary and reactivation models will help to define the key host parameters that control the infections and are important for the research and development of new therapeutic and vaccine strategies against cryptococcosis.

Antifungal Drug Development: Targeting the Fungal Sphingolipid Pathway.

Fungal infections are becoming more prevalent and problematic due to the continual rise of immune deficient patients as well as the progressive development of drug resistance towards currently available antifungal drugs. There has been a significant increase in the development of antifungal compounds with a similar mechanism of action of current drugs. In contrast, there has been very little progress in developing compounds inhibiting totally new fungal targets or/and fungal pathways. This review focuses on novel compounds recently discovered to target the fungal sphingolipids and their metabolizing enzymes.

Steryl Glycosides in Fungal Pathogenesis: An Understudied Immunomodulatory Adjuvant.

Invasive fungal infections pose an increasing threat to human hosts, especially in immunocompromised individuals. In response to the increasing morbidity and mortality of fungal infections, numerous groups have shown great strides in uncovering novel treatment options and potential efficacious vaccine candidates for this increasing threat due to the increase in current antifungal resistance. Steryl glycosides are lipid compounds produced by a wide range of organisms, and are largely understudied in the field of pathogenicity, especially to fungal infections. Published works over the years have shown these compounds positively modulating the host immune response. Recent advances, most notably from our lab, have strongly indicated that steryl glycosides have high efficacy in protecting the host against lethal Cryptococcal infection through acting as an immunoadjuvant. This review will summarize the keystone studies on the role of steryl glycosides in the host immune response, as well as elucidate the remaining unknown characteristics and future perspectives of these compounds for the host-fungal interactions.