Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages

The fungal pathogen Histoplasma capsulatum (Hc) invades, replicates within, and destroys macrophages. To interrogate the molecular mechanisms underlying this interaction, we conducted a host-directed CRISPR-Cas9 screen and identified 361 genes that modify macrophage susceptibility to Hc infection, greatly expanding our understanding of host gene networks targeted by Hc. We identified pathways that have not been previously implicated in Hc interaction with macrophages, including the ragulator complex (involved in nutrient stress sensing), glycosylation enzymes, protein degradation machinery, mitochondrial respiration genes, solute transporters, and the ER membrane complex (EMC). The highest scoring protective hits included the complement C3a receptor (C3aR), a G-protein coupled receptor (GPCR) that recognizes the complement fragment C3a. Although it is known that the complement system reacts with the fungal surface, leading to opsonization and release of small peptide fragments such as C3a, a role for C3aR in macrophage susceptibility to fungi has not been elucidated. We demonstrated that whereas C3aR is dispensable for macrophage phagocytosis of bacteria and latex beads, it is critical for optimal macrophage capture of pathogenic fungi, including Hc,the ubiquitous fungal pathogen Candida albicans, and the causative agent of Valley Fever Coccidioides posadasii. We showed that C3aR localizes to the early phagosome during H. capsulatum infection where it coordinates the formation of actin-rich membrane protrusions that promote Hc capture. We also showed that the EMC promotes surface expression of C3aR, likely explaining its identification in our screen. Taken together, our results provide new insight into host processes that affect Hc-macrophage interactions and uncover a novel and specific role for C3aR in macrophage recognition of fungi.

Primary Central Nervous System Infection by Histoplasma in an Immunocompetent Adult: A Case Report

Histoplasma capsulatum infection is endemic in many regions around the world, including Latin America [1]. However, cerebral presentation occurs in less than 25% of patients with disseminated histoplasmosis and even rarer as a stand-alone presentation. Three forms are described: meningeal, miliary granulomatous and parenchymal with formation of “histoplasmoma” [2]. Due to the rarity of the case and unusual clinical presentation and topography we describe the case below.

Factores relacionados con histoplasmosis diseminada en los pacientes admitidos con VIH-SIDA y fiebre, CHDrAAM, 2017 - 2019

Introducción: el diagnóstico de histoplasmosis diseminada (HD) es un desafío debido a la similitud del cuadro clínico con otras enfermedades infecciosas y no infecciosas. Confirmar un caso de HD requiere realizar procedimientos poco sensibles como hemocultivos, mielocultivos y biopsias de tejidos, o costosos como el antígeno urinario de Histoplasma capsulatum. Conocer los factores clínicos y de laboratorios relacionados con el diagnóstico de HD es de vital importancia en especial en sitios con recursos limitados. Metodología: se realizó un estudio de cohorte retrospectivo donde los expedientes clínicos de los pacientes con fiebre y VIH-SIDA, admitidos con sospecha de histoplasmosis diseminada del 1 de enero de 2017 al 31 de diciembre de 2019 en el Complejo Hospitalario Dr. Arnulfo Arias Madrid (CHDrAAM) de Panamá, fueron evaluados por características demográficas, clínicas y de laboratorio relacionadas con el diagnóstico de HD. Resultados: 63 pacientes cumplieron los criterios de inclusión, 14 con diagnóstico confirmado de HD y 49 con diagnóstico no confirmado de HD. Los niveles de lactato deshidrogenasa (LDH) y aspartato transaminasa (AST) se relacionaron con el diagnóstico de HD en el análisis univariado. Luego de realizar regresión logística ni AST ni LDH fueron estadísticamente significativas. (p= 0.22 y P = 0.92). Conclusión: Ninguna característica clínica ni de laboratorio se relacionó de forma independiente con el diagnóstico de HD, en pacientes con VIH-SIDA admitidos con sospecha de histoplasmosis diseminada.

Mystery of Mesenteric Lymph Adenitis

The etiological factors are confusing for provisional diagnosis and the differential diagnosis of mesenteric lymph adenitis; it may be virus like Dengue, Herpes and Epstein - Barr virus. Bacterial infections like Tuberculosis of the intestine through contaminated unpasteurized cattle milk or Mycobacterium tuberculosis through infected swallowed sputum. T. Gondii, Yesinia enterocolitica, pseudo tuberculosis infection. Lupus vulgaris in the face at the mucocutaneous junctions is a reactivation of already existing tuberculosis facilitates clinical diagnosis. Fungal infections like mucor mycosis, aspergillus, Fusarium producing neutropenia, Histoplasma capsulatum, Cryptococcus aerosol infection from droppings of pigeons on the AC machines. Kikuchi-Fujimoto’s disease (KFD) . Autoimmune causative factors. A study to reveal the mystery on patients with above symptoms and signs to rule out infections or a complication of follicular lymphoma.

Coinfecção entre HIV e Histoplasma capsulatum

A histoplasmose é uma doença endêmica causada por Histoplasma capsulatum. A coinfecção entre HIV e H. capsulatum faz com que os pacientes estejam sob alto risco de desenvolver a forma disseminada da doença com alta letalidade. O objetivo deste estudo foi realizar um levantamento contendo as últimas descobertas ou pesquisas conduzidas nos últimos anos sobre a coinfecção. Trata-se de uma revisão integrativa, onde foram utilizadas as plataformas PubMed, SciELO e LILACS. Foram cruzados com o operador booleano OR e AND os descritores: “HIV”, “AIDS”, “Coinfection”, “Histoplasma” e “Histoplasmosis” em inglês, português e espanhol. Os critérios de inclusão foram: artigos na íntegra publicados entre 2016 a 2021. Os critérios de exclusão foram: artigos duplicados, resenhas, anais de congresso, artigos de revisão, opinião ou reflexão, editoriais, artigos que não abordaram o tema e publicados fora do período de análise. Foram selecionados 25 artigos. Em relação à temática dos artigos, 68% são estudos sobre a prevalência/incidência da coinfecção, 20% tratam das manifestações clínicas, 8% são estudos de métodos diagnósticos e 4% tratam da diversidade genética de Histoplasma encontrada em pacientes HIV. Não houve nenhum estudo sobre tratamento da coinfecção. Quanto a origem, 68% dos artigos são de países da América Latina. Esta revisão possibilitou traçar um panorama sobre os últimos estudos publicados a respeito da coinfecção. Percebe-se a ausência de estudos com enfoque no tratamento e faz-se necessário intensificar pesquisas neste aspecto e realizar inquéritos sorológicos para determinar a prevalência real da Histoplasmose pois ainda é uma doença relacionada à AIDS amplamente subdiagnosticada.

Epidemiology of Epizootic Lymphangitis Among Carthorses in Ethiopia

Epizootic lymphangitis caused by Histoplasma capsulatum variety farciminosum is a debilitating disease incurring considerable economic losses and affecting the welfare of carthorses. Understanding of its epidemiology is important for devising effective prevention and control measures. A cross-sectional study was conducted on 4,162 carthorses in 17 towns in Ethiopia between October 2018 and June 2019. Clinical and microscopic examinations, fungal culturing, and polymerase chain reaction were used to undertake this study. The overall prevalence of epizootic lymphangitis was 16.67% (95% CI: 15.55–17.84) in carthorses. Epizootic lymphangitis was detected in carthorses found in 16 of the 17 towns included in the study. The highest prevalence was recorded at Kombolcha Town (33.33; 95% CI: 27.54–39.52) whereas the lowest was recorded at Debre Birhan Town (0.00; 95% CI: 0.00–1.27). The results of univariable firth logistic regression analysis showed that the difference between the prevalence of Kombolcha and the prevalences of all the other towns except Holota and Shashemene were statistically significant. Statistically significantly lower prevalence was observed in other towns. Classification of the cases into different clinical forms showed that 87.18, 4.33, and 0.58% were cutaneous, ocular, and respiratory forms respectively, while the remaining 7.93% (55/694; 95% CI: 6.03–10.19) were classified as mixed form. In terms of the severity of the disease, 28.67, 60.52, and 0.81% were mild, moderate, and severe cases, respectively. The majority of the lesions (43.95%) were observed in the skin followed by forelimbs (14.55%) and neck region (14.27%). Higher mean annual temperature, lower annual rainfall, and higher humidity of the study towns were statistically significantly associated with an increased risk of epizootic lymphangitis. In conclusion this study revealed widespread occurrence of epizootic lymphangitis in carthorses yet a heterogeneous prevalence between towns. The veterinary and livestock authorities should take this into account while devising disease control.

A Histoplasma capsulatum Lipid Metabolic Map Identifies Antifungal Targets

It is estimated that 150 people die per hour due to the insufficient therapeutic treatments to combat fungal infections. A major hurdle to developing antifungal therapies is the scarce knowledge on the fungal metabolic pathways and mechanisms of virulence.

Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

MANIFESTAÇÃO ATÍPICA DE HISTOPLASMOSE EM TERCEIRA PÁLPEBRA DE GATO DOMÉSTICO – RELATO DE CASO

Introdução: o Histoplasma capsulatum é um fungo dimórfico oportunista causador da histoplasmose, uma micose zoonótica de grande importância. A contaminação ocorre via inalação e os sinais dependem do órgão acometido, mas com apresentação mais comum cutânea e/ou respiratória. A manifestação ocular é considerada rara e pouco descrita na literatura. Objetivo: relatar um caso atípico de histoplasmose na terceira pálpebra de um gato. Material e Métodos: foi atendido um gato, fêmea, sem raça definida, 12 anos, devido a quemose em olho direito (OD) e hiporexia. O animal era portador do vírus da leucemia felina (FeLV). O paciente foi previamente medicado com corticoide, com piora da quemose posterior a suspensão. Ao exame oftálmico, observou-se terceira pálpebra com presença de edema, irregularidade e úlcera, além de hiperemia conjuntival, secreções e crostas no OD. O paciente foi submetido ao exame citológico coletado por aspiração com agulha fina da terceira pálpebra apresentando resultado sugestivo de processo inflamatório crônico-ativo. O paciente foi submetido a coleta de exames hematológicos, radiografia torácica, ultrassonografia abdominal e repetição da citologia. Não foram detectadas presença de neoformações nos exames de imagem e a citologia apresentou o mesmo resultado da citologia anterior. Procedeu-se a excisão cirúrgica de terceira pálpebra com envio do material para histopatológico. Resultados: nos exames sanguíneos, foi detectada neutrofilia, trombocitopenia e marcadores hepáticos e renais elevados, o que corrobora os achados ultrassonográficos que detectou nefropatia crônica bilateral e esteatose hepática. A análise histopatológica, revelou intenso infiltrado inflamatório com histiócitos epitelióides difusos, linfócitos, plasmócitos e neutrófilos, presença de inúmeras estruturas diminutas localizadas no citoplasma desses histiócitos, núcleo redondo e citoplasma claro, compatível com inflamação piogranulomatosa com presença de H. capsulatum, diagnosticando a histoplasmose. Conclusão: sugere-se que a infecção tenha ocorrido devido a imunossupressão do animal FeLV positivo, pelo caráter oportunista da micose. Sendo importante incluir este agente H. capsulatum como diagnóstico diferencial para neoformações em terceira pálpebra de gatos. Além disso, reafirma-se a importância do exame histopatológico como diagnóstico definitivo de lesões, mesmo em tecidos atípicos para o agente.

Deletion of the Stress Response Gene DDR48 from Histoplasma capsulatum Increases Sensitivity to Oxidative Stress, Increases Susceptibility to Antifungals, and Decreases Fitness in Macrophages

The stress response gene DDR48 has been characterized in Saccharomyces cerevisiae and Candida albicans to be involved in combating various cellular stressors, from oxidative agents to antifungal compounds. Surprisingly, the biological function of DDR48 has yet to be identified, though it is likely an important part of the stress response. To gain insight into its function, we characterized DDR48 in the dimorphic fungal pathogen Histoplasma capsulatum. Transcriptional analyses showed preferential expression of DDR48 in the mycelial phase. Induction of DDR48 in Histoplasma yeasts developed after treatment with various cellular stress compounds. We generated a ddr48∆ deletion mutant to further characterize DDR48 function. Loss of DDR48 alters the transcriptional profile of the oxidative stress response and membrane synthesis pathways. Treatment with ROS or antifungal compounds reduced survival of ddr48∆ yeasts compared to controls, consistent with an aberrant cellular stress response. In addition, we infected RAW 264.7 macrophages with DDR48-expressing and ddr48∆ yeasts and observed a 50% decrease in recovery of ddr48∆ yeasts compared to wild-type yeasts. Loss of DDR48 function results in numerous negative effects in Histoplasma yeasts, highlighting its role as a key player in the global sensing and response to cellular stress by fungi.