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Cryptococcosis, caused by fungi of the genus Cryptococcus, manifests in a broad range of clinical presentations, including severe pneumonia and disease of the central nervous system (CNS) and other tissues (bone and skin). Immune deficiency or development of overexuberant inflammatory responses can result in increased susceptibility or host damage, respectively, during fungal encounters. Leukotrienes help regulate inflammatory responses against fungal infections. Nevertheless, studies showed that Cryptococcus exploits host 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, to facilitate transmigration across the brain-blood barrier. To investigate the impact of host 5-LO on the development of protective host immune responses and mortality during cryptococcosis, wild-type (C57BL/6) and 5-lipoxygenase-deficient (5-LO-/-) mice were given experimental pulmonary and systemic Cryptococcus sp., infections. Our results showed that 5-LO-/- mice exhibited reduced pathology and better disease outcomes (i.e., no mortality or signs associated with cryptococcal meningoencephalitis) following pulmonary infection with C. deneoformans, despite having detectable yeast in the brain tissues. In contrast, C57BL/6 mice exhibited classical signs associated with cryptococcal meningoencephalitis. Additionally, brain tissues of 5-LO-/- mice exhibited lower levels of cytokines (CCL2 and CCL3) clinically associated with Cryptococcus-related immune reconstitution inflammatory syndrome (C-IRIS). In a systemic mouse model of cryptococcosis, 5-LO-/- mice and those treated with a Federal Drug Administration (FDA)-approved 5-LO synthesis inhibitor, zileuton, displayed significantly reduced mortality compared to C57BL/6 infected mice. These results suggest that therapeutics designed to inhibit host 5-LO signaling could reduce disease pathology and mortality associated with cryptococcal meningoencephalitis. IMPORTANCE: Cryptococcosis is a mycosis with worldwide distribution and has a broad range of clinical manifestations, including diseases of the CNS. Globally, there is an estimated 179,000 cases of cryptococcal meningitis, resulting in approximately 112,000 fatalities per annum and 19% of AIDS-related deaths. Understanding how host immune responses are modulated during cryptococcosis is central to mitigating the morbidity and mortality associated with cryptococcosis. Leukotrienes (LTs) have been shown to modulate inflammatory responses during infection. In this study, we show that mice deficient in 5-lipoxygenase (5-LO), an enzyme central to the metabolism of arachidonic acid into leukotrienes, exhibit reduced pathology, disease, and neurological signs associated with cryptococcal meningitis. Additionally, mice given an experimental cryptococcal infection and subsequently treated with an FDA-approved 5-LO synthesis inhibitor exhibited significantly reduced mortality rates. These results suggest that therapeutics designed to inhibit host 5-LO activity could significantly reduce pathology and mortality rates associated with cryptococcal meningitis.
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Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.
Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Animais , Criptococose/microbiologia , Criptococose/tratamento farmacológico , Criptococose/imunologia , Camundongos , Cryptococcus neoformans/patogenicidade , Cryptococcus gattii/patogenicidade , Antifúngicos/farmacologia , Antifúngicos/uso terapêuticoRESUMO
Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen found ubiquitously in the environment that causes pneumonia and life-threatening infections of the central nervous system. Following inhalation of yeasts or desiccated basidiospores into the lung alveoli, resident pulmonary phagocytic cells aid in the identification and eradication of Cryptococcus yeast through their arsenal of pattern recognition receptors (PRRs). PRRs recognize conserved pathogen-associated molecular patterns (PAMPs), such as branched mannans, ß-glucans, and chitins that are the major components of the fungal cell wall. However, the key receptors/ligand interactions required for cryptococcal recognition and eventual fungal clearance have yet to be elucidated. Here we present an imaging flow cytometer (IFC) method that offers a novel quantitative cellular imaging and population statistics tool to accurately measure phagocytosis of fungal cells. It has the capacity to measure two distinct steps of phagocytosis: association/attachment and internalization in a high-throughput and quantitative manner that is difficult to achieve with other technologies. Results from these IFC studies allow for the potential to identify PRRs required for recognition, uptake, and subsequent activation of cytokine production, as well as other effector cell responses required for fungal clearance.
Assuntos
Cryptococcus neoformans , Citometria de Fluxo , Fagocitose , Citometria de Fluxo/métodos , Cryptococcus neoformans/metabolismo , Animais , Camundongos , Fagócitos/metabolismo , Fagócitos/microbiologia , Criptococose/microbiologia , Criptococose/metabolismo , Criptococose/imunologia , Cryptococcus/metabolismo , Humanos , Citometria por Imagem/métodos , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Candidiasis is one of the most frequent nosocomial infections affecting an increasing number of at-risk patients. Candida albicans remains the most frequent causative agent of candidiasis, but, in the last decade, C. auris has emerged as a formidable multi-drug-resistant pathogen. Both species are fully capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. Here, we have screened the Repurposing Hub library from the Broad Institute, containing over 6000 compounds, in search for inhibitors of C. albicans and C. auris biofilm formation. The primary screen identified 57 initial hits against C. albicans and 33 against C. auris. Confirmatory concentration-dependent assays were used to validate the activity of the initial hits and, at the same time, establish their anti-biofilm potency. Based on these results, ebselen, temsirolimus, and compound BAY 11-7082 emerged as the leading repositionable compounds. Subsequent experiments established their spectrum of antifungal activity against yeasts and filamentous fungi. In addition, their in vivo activity was examined in the murine models of hematogenously disseminated C. albicans and C. auris infections. Although promising, further in vitro and in vivo studies are needed to confirm their potential use for the therapy of candidiasis and possibly other fungal infections.
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Candida spp. are opportunistic yeasts capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. We screened the Pandemic Response Box containing 400 diverse drug-like molecules active against bacteria, viruses or fungi, for inhibitors of Candida albicans and Candida auris biofilm formation. Initial hits were identified based on the demonstration of >70% inhibitory activity. Dose-response assays were used to confirm the antifungal activity of initial hits and establish their potency. The spectrum of antifungal activity of the leading compounds was determined against a panel of medically important fungi, and the in vivo activity of the leading repositionable agent was evaluated in murine models of C. albicans and C. auris systemic candidiasis. The primary screening identified 20 hit compounds, and their antifungal activity and potency against C. albicans and C. auris were validated using dose-response measurements. From these experiments, the rapalog everolimus, emerged as the leading repositionable candidate. Everolimus displayed potent antifungal activity against different Candida spp., but more moderate levels of activity against filamentous fungi. Treatment with everolimus increased survival of mice infected with C. albicans, but not those with C. auris. The screening of the Pandemic Response Box resulted in the identification of several drugs with novel antifungal activity, with everolimus emerging as the main repositionable candidate. Further in vitro and in vivo studies are needed to confirm its potential therapeutic use.
Assuntos
Antifúngicos , Candida albicans , Camundongos , Animais , Candida albicans/fisiologia , Antifúngicos/farmacologia , Candida auris , Everolimo/farmacologia , Pandemias , Candida , Biofilmes , Testes de Sensibilidade MicrobianaRESUMO
Vaccines are one of the most effective public health tools to prevent and manage infectious diseases. Since the first clinical use of vaccines in the late 18th century, many vaccines have been successfully developed to combat bacterial and viral infections, including the most recent Coronavirus Disease 2019 (COVID-19) pandemic. However, there remains no vaccine that is clinically available to treat or prevent invasive fungal diseases, including cryptococcal meningoencephalitis. This fungal disease is uniformly fatal without treatment and has a global mortality rate of over 70%. Despite a dire need for an effective cryptococcal vaccine, there are many scientific and economic challenges to overcome prior to making it a reality. Here, we discuss some of these challenges as well as steps that the community is taking for commercialization of effective cryptococcal vaccines.
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COVID-19 , Doenças Transmissíveis , Cryptococcus neoformans , Micoses , Vacinas , Vacinas Virais , HumanosRESUMO
Many successful pathogens cause latent infections, remaining dormant within the host for years but retaining the ability to reactivate to cause symptomatic disease. The human opportunistic fungal pathogen Cryptococcus neoformans establishes latent pulmonary infections in immunocompetent individuals upon inhalation from the environment. These latent infections are frequently characterized by granulomas, or foci of chronic inflammation, that contain dormant and persistent cryptococcal cells. Immunosuppression can cause these granulomas to break down and release fungal cells that proliferate, disseminate, and eventually cause lethal cryptococcosis. This course of fungal latency and reactivation is understudied due to limited models, as chronic pulmonary granulomas do not typically form in mouse cryptococcal infections. A loss-of-function mutation in the Cryptococcus-specific MAR1 gene was previously described to alter cell surface remodeling in response to host signals. Here, we demonstrate that the mar1Δ mutant strain persists long term in a murine inhalation model of cryptococcosis, inducing a chronic pulmonary granulomatous response. We find that murine infections with the mar1Δ mutant strain are characterized by reduced fungal burden, likely due to the low growth rate of the mar1Δ mutant strain at physiological temperature, and an altered host immune response, likely due to inability of the mar1Δ mutant strain to properly employ virulence factors. We propose that this combination of features in the mar1Δ mutant strain collectively promotes the induction of a more chronic inflammatory response and enables long-term fungal persistence within these granulomatous regions.
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Criptococose , Cryptococcus neoformans , Infecção Latente , Animais , Criptococose/microbiologia , Modelos Animais de Doenças , Inflamação , Pulmão , CamundongosRESUMO
Recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen is the hallmark of an acute immunopathologic inflammatory response during vulvovaginal candidiasis (VVC) caused by Candida albicans. Recurrent VVC (RVVC) remains a chronic health burden in affected women worldwide despite the use of antifungal therapy. Based on the role leukotrienes (LTs) play in promoting inflammation, leukotriene receptor antagonists (LTRAs) targeted for LTB4 (etalocib) or LTC4, LTD4, and LTE4 (zafirlukast or montelukast) have been shown to reduce inflammation of epithelial tissues. An open-label pilot study using long-term regimens of zafirlukast in women with RVVC indicated the potential for some relief from recurrent episodes. To investigate this clinical observation further, we evaluated the effects of LT antagonistic agents and LT deficiency on the immunopathogenic response in a mouse model of VVC. Results showed that mice given daily intraperitoneal injections of individual LTRAs, starting 2days prior to vaginal inoculation with C. albicans and continuing through 14days post-inoculation, had no measurable reduction in PMN migration. The LTRAs were also ineffective in reducing levels of the hallmark vaginal inflammatory markers (S100A8, IL-1ß) and tissue damage (LDH) associated with the immunopathogenic response. Finally, LT-deficient 5-lipoxygenase knockout mice showed comparable levels of vaginal fungal burden and PMN infiltration to wild-type mice following inoculation with a vaginal (ATCC 96113) or laboratory (SC5314) C. albicans isolate. These results indicate that despite some clinical evidence suggestive of off-target efficacy of LTRAs in RVVC, LTs and associated signaling pathways appear to be dispensable in the immunopathogenesis of VVC.
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The way that diversity, equity, and inclusion impact scientific careers varies for everyone, but it is evident that institutions providing an environment where being different or having differences creates a sense of being welcomed, supported, and valued are beneficial to the scientific community at large. In this commentary, three short stories from Texas-based microbiologists are used to depict (i) the importance of bringing the guiding principles of diversity, equity, and inclusion within their professional roles, (ii) the need to apply and translate those principles to support and enable successful scientific careers among peers and trainees, and (iii) the impact of effective science communication to increase the understanding of microbial environments among the community at large.
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Diversidade Cultural , Microbiologia , Emprego , Humanos , TexasRESUMO
Ocular HSV-1 infection is a major cause of eye disease and innate and adaptive immunity both play a role in protection and pathology associated with ocular infection. Previously we have shown that M1-type macrophages are the major and earliest infiltrates into the cornea of infected mice. We also showed that HSV-1 infectivity in the presence and absence of M2-macrophages was similar to wild-type (WT) control mice. However, it is not clear whether the absence of M1 macrophages plays a role in protection and disease in HSV-1 infected mice. To explore the role of M1 macrophages in HSV-1 infection, we used mice lacking M1 activation (M1-/- mice). Our results showed that macrophages from M1-/- mice were more susceptible to HSV-1 infection in vitro than were macrophages from WT mice. M1-/- mice were highly susceptible to ocular infection with virulent HSV-1 strain McKrae, while WT mice were refractory to infection. In addition, M1-/- mice had higher virus titers in the eyes than did WT mice. Adoptive transfer of M1 macrophages from WT mice to M1-/- mice reduced death and rescued virus replication in the eyes of infected mice. Infection of M1-/- mice with avirulent HSV-1 strain KOS also increased ocular virus replication and eye disease but did not affect latency-reactivation seen in WT control mice. Severity of virus replication and eye disease correlated with significantly higher inflammatory responses leading to a cytokine storm in the eyes of M1-/- infected mice that was not seen in WT mice. Thus, for the first time, our study illustrates the importance of M1 macrophages specifically in primary HSV-1 infection, eye disease, and survival but not in latency-reactivation.
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Síndrome da Liberação de Citocina/imunologia , Ceratite Herpética/imunologia , Macrófagos/imunologia , Animais , Herpesvirus Humano 1/imunologia , Camundongos , Ativação Viral/imunologia , Latência Viral/imunologiaRESUMO
The Peptidoglycan (PG) cell wall of the Lyme disease (LD) spirochete, Borrelia burgdorferi (Bb), contributes to structural and morphological integrity of Bb; is a persistent antigen in LD patients; and has a unique pentapeptide with L-Ornithine as the third amino acid that cross-links its glycan polymers. A borrelial homolog (BB_0167) interacted specifically with borrelilal PG via its peptidoglycan interacting motif (MHELSEKRARAIGNYL); was localized to the protoplasmic cylinder of Bb; and was designated as Borrelia peptidoglycan interacting Protein (BpiP). A bpiP mutant displayed no defect under in vitro growth conditions with similar levels of several virulence-related proteins. However, the burden of bpiP mutant in C3H/HeN mice at day 14, 28 and 62 post-infection was significantly lower compared to control strains. No viable bpiP mutant was re-isolated from any tissues at day 62 post-infection although bpiP mutant was able to colonize immunodeficient SCID at day 28 post-infection. Acquisition or transmission of bpiP mutant by Ixodes scapularis larvae or nymphs respectively, from and to mice, was significantly lower compared to control strains. Further analysis of bpiP mutant revealed increased sensitivity to vancomycin, osmotic stress, lysosomal extracts, human antimicrobial peptide cathelicidin-LL37, complement-dependent killing in the presence of day 14 post-infection mouse serum and increased internalization of CFSC-labeled bpiP mutant by macrophages and dendritic cells compared to control strains. These studies demonstrate the importance of accessory protein/s involved in sustaining integrity of PG and cell envelope during different phases of Bb infection.
Assuntos
Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/patogenicidade , Interações Hospedeiro-Patógeno , Doença de Lyme , Animais , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Aptidão Genética/fisiologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Fatores Imunológicos/fisiologia , Doença de Lyme/genética , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Doença de Lyme/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos SCID , Peptidoglicano/metabolismo , Virulência/genéticaRESUMO
Caspase recruitment domain-containing protein 9 (CARD9) is a critical adaptor molecule triggered by the interaction of C-type lectin receptors (CLRs) with carbohydrate motifs found in fungi. Consequently, clinical and animal studies indicate that CARD9 is an important regulator of protective immunity against fungal pathogens. Previous studies suggest that CARD9 is important for the induction of protection against Cryptococcus neoformans, an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system in immunocompromised patients. However, the effect of CARD9 deficiency on the induction of protective immune responses against C. neoformans is unknown. Immunization with a C. neoformans mutant that overexpresses the transcription factor zinc finger 2, denoted LW10, results in protection against an otherwise lethal challenge with wild-type (WT) C. neoformans Our results showed that CARD9 is essential for the induction of vaccine-mediated immunity against C. neoformans infection. We observed significant decreases in interleukin-17 (IL-17) production and significant increases in Th2-type cytokine (IL-4, IL-5, and IL-13) production in CARD9-deficient mice after inoculation with strain LW10. While leukocyte infiltration to the lungs of CARD9-deficient mice was similar in LW10 and WT C. neoformans-infected mice, macrophages derived from CARD9-deficient mice inherently skewed toward an M2 activation phenotype, were unable to contain the growth of LW10, and failed to produce nitric oxide in response to infection with LW10 or stimulation with lipopolysaccharide. These results suggest that CARD9-mediated signaling is required for M1 macrophage activation and fungicidal activity necessary for the induction of vaccine-mediated immunity against C. neoformansIMPORTANCECryptococcus neoformans is a fungal pathogen that is found throughout the environment and can cause life-threatening infections of the lung and central nervous system in severely immunocompromised individuals. Caspase recruitment domain-containing protein 9 (CARD9) is a critical molecule that is activated after interactions of C-type lectin receptors (CLRs) found on the surfaces of specific immune cells, with carbohydrate structures associated with fungi. Patients with defects in CARD9 are significantly more susceptible to a multitude of fungal infections. C. neoformans contains several carbohydrate structures that interact with CLRs on immune cells and activate CARD9. Consequently, these studies evaluated the necessity of CARD9 for the induction of protective immunity against C. neoformans infection. These results are important, as they advance our understanding of cryptococcal pathogenesis and host factors necessary for the induction of protective immunity against C. neoformans.
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Proteínas Adaptadoras de Sinalização CARD/genética , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus neoformans/imunologia , Pneumopatias Fúngicas/imunologia , Pneumopatias Fúngicas/microbiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Animais , Biomarcadores , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Criptococose/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Resistência à Doença/imunologia , Feminino , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunofenotipagem , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Pneumopatias Fúngicas/metabolismo , Masculino , Camundongos , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
Arrestins, a structurally specialized and functionally diverse group of proteins, are central regulators of adaptive cellular responses in eukaryotes. Previous studies on fungal arrestins have demonstrated their capacity to modulate diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. However, the mechanisms by which arrestin-regulated processes are involved in fungal virulence remain unexplored. We have identified a small family of four arrestins, Ali1, Ali2, Ali3, and Ali4, in the human fungal pathogen Cryptococcus neoformans Using complementary microscopy, proteomic, and reverse genetics techniques, we have defined a role for Ali1 as a novel contributor to cytokinesis, a fundamental cell cycle-associated process. We observed that Ali1 strongly interacts with proteins involved in lipid synthesis, and that ali1Δ mutant phenotypes are rescued by supplementation with lipid precursors that are used to build cellular membranes. From these data, we hypothesize that Ali1 contributes to cytokinesis by serving as an adaptor protein, facilitating the localization of enzymes that modify the plasma membrane during cell division, specifically the fatty acid synthases Fas1 and Fas2. Finally, we assessed the contributions of the C. neoformans arrestin family to virulence to better understand the mechanisms by which arrestin-regulated adaptive cellular responses influence fungal infection. We observed that the C. neoformans arrestin family contributes to virulence, and that the individual arrestin proteins likely fulfill distinct functions that are important for disease progression.IMPORTANCE To survive under unpredictable conditions, all organisms must adapt to stressors by regulating adaptive cellular responses. Arrestin proteins are conserved regulators of adaptive cellular responses in eukaryotes. Studies that have been limited to mammals and model fungi have demonstrated that the disruption of arrestin-regulated pathways is detrimental for viability. The human fungal pathogen Cryptococcus neoformans causes more than 180,000 infection-related deaths annually, especially among immunocompromised patients. In addition to being genetically tractable, C. neoformans has a small arrestin family of four members, lending itself to a comprehensive characterization of its arrestin family. This study serves as a functional analysis of arrestins in a pathogen, particularly in the context of fungal fitness and virulence. We investigate the functions of one arrestin protein, Ali1, and define its novel contributions to cytokinesis. We additionally explore the virulence contributions of the C. neoformans arrestin family and find that they contribute to disease establishment and progression.
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Arrestina/metabolismo , Ciclo Celular , Suscetibilidade a Doenças , Fungos/fisiologia , Micoses/microbiologia , Arrestina/genética , Biomarcadores , Ciclo Celular/genética , Citocinese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metabolismo dos Lipídeos , Modelos Biológicos , Mutação , Micoses/metabolismo , Virulência , Proteínas ras/metabolismoRESUMO
BACKGROUND: Cryptococcus neoformans, the causative agent of cryptococcosis, causes ~ 181,000 deaths annually, with males having a higher incidence of disease than females (7M:3F). The reason for this sex bias remains unclear. We hypothesized that this disparity was due to biological differences between the male and female immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and infected with C. neoformans ± exogenous testosterone or 17-ß-estradiol. C. neoformans, B, T, and NK cell proliferation was quantified by flow cytometry. Cytokine analysis was conducted via protein array or ELISA. Serological testing was conducted to determine previous exposure to C. neoformans. RESULTS: C. neoformans proliferated more in male PBMCs. T cell percentages in both sexes were lower in infected versus uninfected cells. Male PBMCs had lower CD3+, CD4+, and CD8+ T cells percentages during infection compared to females. Cytokine profiles showed differences in uninfected male and female PBMCs, which subsided during infection. Only one donor was sero-negative for prior C. neoformans exposure. There was an effect of estrogen in one dataset. CONCLUSIONS: These results suggest that males show an inherent deficit in T cell response during infection, which may contribute to the increased incidence of disease in males.
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Criptococose/imunologia , Cryptococcus neoformans , Leucócitos Mononucleares/microbiologia , Subpopulações de Linfócitos/microbiologia , Adolescente , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Contagem de Linfócitos , Subpopulações de Linfócitos/metabolismo , Masculino , Caracteres SexuaisRESUMO
Dendritic cells (DCs), a vital component of the innate immune system, are considered to lack antigen specificity and be devoid of immunological memory. Strategies that can induce memory-like responses from innate cells can be utilized to elicit protective immunity in immune deficient persons. Here we utilize an experimental immunization strategy to modulate DC inflammatory and memory-like responses against an opportunistic fungal pathogen that causes significant disease in immunocompromised individuals. Our results show that DCs isolated from protectively immunized mice exhibit enhanced transcriptional activation of interferon and immune signaling pathways. We also show long-term memory-like cytokine responses upon subsequent challenge with the fungal pathogen that are abrogated with inhibitors of specific histone modifications. Altogether, our study demonstrates that immunization strategies can be designed to elicit memory-like DC responses against infectious disease.
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Células Dendríticas/imunologia , Memória Imunológica , Animais , Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus/fisiologia , Células Dendríticas/microbiologia , Feminino , Histonas/metabolismo , Imunidade Inata , Inflamação/genética , Inflamação/patologia , Interferon gama/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Camundongos Endogâmicos BALB C , Fenótipo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , VacinaçãoRESUMO
Development of vaccines against opportunistic infections is difficult as patients most at risk of developing disease are deficient in aspects of the adaptive immune system. Here, we utilized an experimental immunization strategy to induce innate memory in macrophages in vivo. Unlike current trained immunity models, we present an innate memory-like phenotype in macrophages that is maintained for at least 70 days post-immunization and results in complete protection against secondary challenge in the absence of adaptive immune cells. RNA-seq analysis of in vivo IFN-γ primed macrophages revealed a rapid up-regulation of IFN-γ and STAT1 signaling pathways following secondary challenge. The enhanced cytokine recall responses appeared to be pathogen-specific, dependent on changes in histone methylation and acetylation, and correlated with increased STAT1 binding to promoter regions of genes associated with protective anti-fungal immunity. Thus, we demonstrate an alternative mechanism to induce macrophage innate memory in vivo that facilitates pathogen-specific vaccine-mediated immune responses.
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Criptococose/prevenção & controle , Cryptococcus neoformans/imunologia , Interferon gama/metabolismo , Pneumopatias Fúngicas/prevenção & controle , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Fator de Transcrição STAT1/metabolismo , Animais , Criptococose/imunologia , Criptococose/microbiologia , Citocinas/metabolismo , Feminino , Pneumopatias Fúngicas/imunologia , Pneumopatias Fúngicas/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Transdução de SinaisRESUMO
Since its original isolation in 2009, Candida auris has spread across the globe as a causative agent of invasive candidiasis. C. auris is typically intrinsically resistant to fluconazole and can also be resistant to echinocandins and even amphotericin B. Thus, there is an urgent need to find new treatment options against this emerging pathogen. To address this growing problem, we performed a screen of the Prestwick Chemical library, a repurposing library of 1,280 small molecules, consisting mostly of approved off-patent drugs, in search of those with activity against a multidrug-resistant C. auris isolate. Our initial screen, using standardized susceptibility testing methodologies, identified nine miscellaneous compounds with no previous clinical indication as antifungals or antiseptics that displayed activity against C. auris Confirmation and follow-up studies identified ebselen as the drug displaying the most potent activity, with 100% inhibition of growth detected at concentrations as low as 2.5 µM. We further evaluated the ability of ebselen to inhibit C. auris biofilm formation and examined the effects of combination therapies of ebselen with clinically used antifungals. We extended our studies to different C. auris strains with various susceptibility patterns and also confirmed its antifungal activity against Candida albicans and clinical isolates of multiple other Candida species. Furthermore, ebselen displayed a broad spectrum of antifungal actions on the basis of its activity against a variety of medically important fungi, including yeasts and molds. Overall, our results indicate the promise of ebselen as a repositionable agent for the treatment of candidiasis and possibly other mycoses and, in particular, for the treatment of infections refractory to conventional treatment with current antifungals.
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Antifúngicos/farmacologia , Azóis/farmacologia , Candida/efeitos dos fármacos , Reposicionamento de Medicamentos/métodos , Compostos Organosselênicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/metabolismo , Farmacorresistência Fúngica Múltipla , IsoindóisRESUMO
The human fungal pathogen, Cryptococcus neoformans, dramatically alters its cell wall, both in size and composition, upon entering the host. This cell wall remodeling is essential for host immune avoidance by this pathogen. In a genetic screen for mutants with changes in their cell wall, we identified a novel protein, Mar1, that controls cell wall organization and immune evasion. Through phenotypic studies of a loss-of-function strain, we have demonstrated that the mar1Δ mutant has an aberrant cell surface and a defect in polysaccharide capsule attachment, resulting in attenuated virulence. Furthermore, the mar1Δ mutant displays increased staining for exposed cell wall chitin and chitosan when the cells are grown in host-like tissue culture conditions. However, HPLC analysis of whole cell walls and RT-PCR analysis of cell wall synthase genes demonstrated that this increased chitin exposure is likely due to decreased levels of glucans and mannans in the outer cell wall layers. We observed that the Mar1 protein differentially localizes to cellular membranes in a condition dependent manner, and we have further shown that the mar1Δ mutant displays defects in intracellular trafficking, resulting in a mislocalization of the ß-glucan synthase catalytic subunit, Fks1. These cell surface changes influence the host-pathogen interaction, resulting in increased macrophage activation to microbial challenge in vitro. We established that several host innate immune signaling proteins are required for the observed macrophage activation, including the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR2 pattern recognition receptors. These studies explore novel mechanisms by which a microbial pathogen regulates its cell surface in response to the host, as well as how dysregulation of this adaptive response leads to defective immune avoidance.
Assuntos
Parede Celular/enzimologia , Criptococose/imunologia , Cryptococcus neoformans/enzimologia , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Evasão da Resposta Imune/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Animais , Parede Celular/imunologia , Células Cultivadas , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus neoformans/patogenicidade , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Proteínas Fúngicas/genética , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transporte Proteico , beta-Glucanas/imunologiaRESUMO
Cryptococcus species, the etiological agents of cryptococcosis, are encapsulated fungal yeasts that predominantly cause disease in immunocompromised individuals, and are responsible for 15% of AIDS-related deaths worldwide. Exposure follows the inhalation of the yeast into the lung alveoli, making it incumbent upon the pattern recognition receptors (PRRs) of pulmonary phagocytes to recognize highly conserved pathogen-associated molecular patterns (PAMPS) of fungi. The main challenges impeding the ability of pulmonary phagocytes to effectively recognize Cryptococcus include the presence of the yeast's large polysaccharide capsule, as well as other cryptococcal virulence factors that mask fungal PAMPs and help Cryptococcus evade detection and subsequent activation of the immune system. This review will highlight key phagocyte cell populations and the arsenal of PRRs present on these cells, such as the Toll-like receptors (TLRs), C-type lectin receptors, NOD-like receptors (NLRs), and soluble receptors. Additionally, we will highlight critical cryptococcal PAMPs involved in the recognition of Cryptococcus. The question remains as to which PRR-ligand interaction is necessary for the recognition, phagocytosis, and subsequent killing of Cryptococcus.
RESUMO
Cryptococcosis remains a significant cause of morbidity and mortality world-wide, particularly among AIDS patients. Yet, to date, there are no licensed vaccines clinically available to treat or prevent cryptococcosis. In this review, we provide a rationale to support continued investment in Cryptococcus vaccine research, potential challenges that must be overcome along the way, and a literature review of the current progress underway towards developing a vaccine to prevent cryptococcosis.