Histoplasma capsulatum requires peroxisomes for multiple virulence functions including siderophore biosynthesis.

Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

A metabolic inhibitor arms macrophages to kill intracellular fungal pathogens by manipulating zinc homeostasis.

Macrophages deploy numerous strategies to combat invasion by microbes. One tactic is to restrict acquisition of diverse nutrients, including trace metals, a process termed nutritional immunity. Intracellular pathogens adapt to a resource-poor environment by marshaling mechanisms to harvest nutrients. Carbon acquisition is crucial for pathogen survival; compounds that reduce availability are a potential strategy to control intracellular replication. Treatment of macrophages with the glucose analog 2-deoxy-D-glucose (2-DG) armed phagocytes to eliminate the intracellular fungal pathogen Histoplasma capsulatum in vitro and in vivo. Killing did not rely on altering access to carbon-containing molecules or changes in ATP, ER stress, or autophagy. Unexpectedly, 2-DG undermined import of exogenous zinc into macrophages, decreasing the quantity of cytosolic and phagosomal zinc. The fungus perished as a result of zinc starvation. This change in metal ingress was not ascribed to a defect in a single importer; rather, there was a collective impairment in transporter activity. This effect promoted the antifungal machinery of macrophages and expanded the complexity of 2-DG activities far beyond manipulating glycolysis. Mechanistic metabolic studies employing 2-DG will have to consider its effect on zinc transport. Our preclinical data support consideration of this agent as a possible adjunctive therapy for histoplasmosis.

Fungal pneumonia in kidney transplant recipients.

Fungal pneumonia is a dreaded complication encountered after kidney transplantation, complicated by increased mortality and often associated with graft failure. Diagnosis can be challenging because the clinical presentation is non-specific and diagnostic tools have limited sensitivity and specificity in kidney transplant recipients and must be interpreted in the context of the clinical setting. Management is difficult due to the increased risk of dissemination and severity, multiple comorbidities, drug interactions and reduced immunosuppression which should be applied as an important adjunct to therapy. This review will focus on the main causes of fungal pneumonia in kidney transplant recipients including Pneumocystis, Aspergillus, Cryptococcus, mucormycetes and Histoplasma. Epidemiology, clinical presentation, laboratory and radiographic features, specific characteristics will be discussed with an update on diagnostic procedures and treatment.

Histoplasmosis.

Histoplasmosis is one of the commonest endemic mycoses in the Americas yet is often underdiagnosed and neglected as a public health priority. This review outlines the evolving understanding of its epidemiology and the clinical syndromes of histoplasmosis, in addition to up-to-date diagnostic and treatment guidelines. A focus on histoplasmosis in advanced HIV is included. The challenges pertinent to histoplasmosis management in Latin America, with recommendations made through international expert consensus are discussed.

Disseminated Histoplasmosis detected on peripheral blood smear examination in immunocompetent patients from non endemic region - Report of two cases from a tertiary care hospital.

Histoplasmosis is an opportunistic systemic infection caused by inhaling spores of a thermal dimorphic fungus Histoplasma capsulatum. Disseminated histoplasmosis is the most common form associated with acquired immune deficiency syndrome (AIDS). However, only a few cases of disseminated histoplasmosis are reported in immuno-competent hosts. Most infections in the immunocompetent hosts are asymptomatic or result in mild pulmonary disease. However the presence of Disseminated Histoplasmosis in immunocompetent host probably results due to prolonged exposure and delayed presentation We report two cases of progressive disseminated histoplasmosis in two immunocompetent patients from non-endemic region in Western Rajasthan, India. Also in both the cases, the first diagnosis was suggested by a peripheral blood smear, which is not a classical biological diagnostic method for fungal infection. Careful examination of Peripheral blood smear along with correct clinical history can aid in early diagnosis of disseminated histoplasmosis even in immunocompetent patients.

Biodiverse Histoplasma Species Elicit Distinct Patterns of Pulmonary Inflammation following Sublethal Infection.

Histoplasma is an endemic dimorphic fungus that can cause disease in healthy and immunocompromised individuals after the transition of inhaled spores into the facultative intracellular yeast form. There is substantial diversity among Histoplasma species, but it is not clear how this heterogeneity impacts the progression of pathology and cellular immune responses during acute respiratory infection, which represents the vast majority of histoplasmosis disease burden. After inoculating mice intranasally with a sublethal inoculum, we characterized the immune response to Histoplasma capsulatum (strain G186A) and Histoplasma ohiense (strain G217B) using comprehensive flow cytometric and single-cell analyses. Within 8 days after inoculation, H. ohiense induced a significantly higher infiltration of neutrophils and inflammatory monocytes into the lung compared to H. capsulatum Microscopic analysis of infected lung tissue revealed that although the total number of fungi was similar within inflamed lung lesions, we observed different species-dependent intracellular yeast distribution patterns. Inoculation with gfp-expressing strains indicated that H. ohiense, but not H. capsulatum, was associated primarily with alveolar macrophages early after infection. Interestingly, we observed a significant reduction in the total number of alveolar macrophages 12 to 16 days after H. ohiense, but not H. capsulatum infection, despite similar intracellular growth dynamics within AMJ2-C11 alveolar macrophages in vitro Together, our data suggest that H. ohiense, but not H. capsulatum, preferentially interacts with alveolar macrophages early after infection, which may lead to a different course of inflammation and resolution despite similar rates of fungal clearance.IMPORTANCE Acute pulmonary histoplasmosis in healthy individuals comprises most of the disease burden caused by the fungal pathogen Histoplasma Fungal pneumonia is frequently delayed in diagnosis and treatment due to a prolonged period of quiescence early during infection. In this study, we used a murine respiratory model of histoplasmosis to investigate how different Histoplasma species modulate lung inflammation throughout the complete course of infection. We propose that a relatively low, sublethal inoculum is ideal to model acute pulmonary histoplasmosis in humans, primarily due to the quiescent stage of fungal growth that occurs in the lungs of mice prior to the initiation of inflammation. Our results reveal the unique course of lung immunity associated with divergent species of Histoplasma and imply that the progression of clinical disease is considerably more heterogeneous than previously recognized.

Bone Marrow Culture Yield for the Diagnosis of Opportunistic Diseases in Patients with AIDS and Disseminated Kaposi Sarcoma.

BACKGROUND: Disseminated Kaposi sarcoma (DKS) is present in patients with advanced HIV infection in whom co-infection with other opportunistic pathogens can occur. Bone marrow (BM) aspirate and biopsy comprise a robust diagnostic tool in patients with fever, cytopenias, and abnormal liver tests. However, the yield in patients with DKS has not been determined. OBJECTIVE: The aim of this study was to evaluate the utility of BM aspirate and biopsy in patients with DKS. METHODS: We included 40 male patients with a recent diagnosis of DKS. BM aspirate and biopsy was performed as part of the workup to rule out co-infections. RESULTS: In four patients, Mycobacterium avium complex (MAC) was recovered from culture. In other four patients, intracellular yeasts were observed in the Grocott stain, diagnosed as Histoplasma. The yield of BM was calculated in 20%. Only 12 patients (30%) had fever and 11 (27.5%) had pancytopenia. Alkaline phosphatase (ALP) above normal values and C-reactive protein (CRP) were higher in patients with positive results for BM than in those with negative results (63% vs. 21.9%, and 3.0 vs. 1.2 mg/L; p = 0.03 in both comparisons). No differences were found when complete blood-count abnormalities were compared. CONCLUSION: We recommend performing a BM aspirate for stains, culture, and biopsy in all HIV patients with DKS, as this will permit the early diagnosis of co-infections and prevent further complications in those who receive chemotherapy.

Cytological diagnosis of primary cutaneous histoplasmosis with hemophagocytosis in immunocompetent patient - A rare case from non endemic region.

Histoplasma capsulatum is an opportunistic dimorphic fungus caused by inhalation of spores present in the soil. It is extremely rare in the northern Himalayan region of India and is usually asymptomatic. It may rarely progress to disseminated histoplasmosis which is usually observed in immunocompromised patients associated with malignancy, acquired immunodeficiency syndrome or diabetes. The present case is being reported because of unusual cutaneous presentation of disseminated histoplasmosis in an immunocompetent patient of non-endemic region. The case also highlights the importance of fine needle aspiration cytology for its precise early diagnosis and avoiding of further complications.

Metabolism of Gluconeogenic Substrates by an Intracellular Fungal Pathogen Circumvents Nutritional Limitations within Macrophages.

Microbial pathogens exploit host nutrients to proliferate and cause disease. Intracellular pathogens, particularly those exclusively living in the phagosome such as Histoplasma capsulatum, must adapt and acquire nutrients within the nutrient-limited phagosomal environment. In this study, we investigated which host nutrients could be utilized by Histoplasma as carbon sources to proliferate within macrophages. Histoplasma yeasts can grow on hexoses and amino acids but not fatty acids as the carbon source in vitro Transcriptional analysis and metabolism profiling showed that Histoplasma yeasts downregulate glycolysis and fatty acid utilization but upregulate gluconeogenesis within macrophages. Depletion of glycolysis or fatty acid utilization pathways does not prevent Histoplasma growth within macrophages or impair virulence in vivo However, loss of function in Pck1, the enzyme catalyzing the first committed step of gluconeogenesis, impairs Histoplasma growth within macrophages and severely attenuates virulence in vivo, indicating that Histoplasma yeasts rely on catabolism of gluconeogenic substrates (e.g., amino acids) to proliferate within macrophages.IMPORTANCEHistoplasma is a primary human fungal pathogen that survives and proliferates within host immune cells, particularly within the macrophage phagosome compartment. The phagosome compartment is a nutrient-limited environment, requiring Histoplasma yeasts to be able to assimilate available carbon sources within the phagosome to meet their nutritional needs. In this study, we showed that Histoplasma yeasts do not utilize fatty acids or hexoses for growth within macrophages. Instead, Histoplasma yeasts consume gluconeogenic substrates to proliferate in macrophages. These findings reveal the phagosome composition from a nutrient standpoint and highlight essential metabolic pathways that are required for a phagosomal pathogen to proliferate in this intracellular environment.

Histoplasmosis Outbreaks in Brazil: Lessons to Learn About Preventing Exposure.

Histoplasmosis is considered the most common invasive opportunistic fungal disease in the Americas, with outbreaks and micro-epidemics reported for over 80 years. In Brazil, this disease has been described since 1946, reaching a remarkable incidence in the population, especially during the HIV-AIDS pandemic. In this study, published and unpublished outbreaks and micro-epidemics of histoplasmosis in Brazil were revisited by accessing different database sources and evaluating epidemiological and clinical features. We have found reports spanning 1946-2017, across 10 Brazilian states and with involvement of 370 humans and 2 dogs, and 13 disseminated cases and 3 deaths were reported. Rio de Janeiro had the largest number of outbreaks (n = 20/40; 50%) reported in this study. The majority of outbreaks and micro-epidemics was reported in caves (n = 21/40; 52.5%), followed by reports in abandoned/deactivated sites (n = 6/40; 15%), mines (n = 5/40; 12.5%), chicken coops (n = 4/40; 10%). Histoplasmosis is a serious health issue in Brazil considering the attractive and growing market of ecotourism throughout more than 7000 caves, and all levels of poultry farming activity are important to raise awareness about how dangerous this neglected disease can be and establish ways to decrease exposure to contaminated environmental sources through adequate preventive measures.

Molecular regulation of Histoplasma dimorphism.

Temperature serves as a fundamental signal in biological systems. In some microbial pathogens of humans, mammalian body temperature triggers establishment and maintenance of a developmental program that allows the microbe to survive and thrive in the host. Histoplasma capsulatum is one of a group of fungal pathogens called thermally dimorphic fungi, all of which respond to mammalian body temperature by converting from an environmental mold form that inhabits the soil into a parasitic form that causes disease in the host. It has been known for decades that temperature is a key signal that is sufficient to trigger the switch from the soil to host form (and vice versa) in the laboratory. Recent molecular studies have identified a number of key regulators that are required to specify each of the developmental forms in response to temperature. Here we review the regulatory circuits that govern temperature-dependent dimorphism in Histoplasma.

Sensing the heat and the host: Virulence determinants of Histoplasma capsulatum.

Histoplasma capsulatum is a member of a group of fungal pathogens called thermally dimorphic fungi, all of which respond to mammalian body temperature by converting from an environmental mold form into a parasitic host form that causes disease. Histoplasma is a primary fungal pathogen, meaning it is able to cause disease in healthy individuals. We are beginning to understand how host temperature is utilized as a key signal to facilitate growth in the parasitic yeast form and promote production of virulence factors. In recent years, multiple regulators of morphology and virulence have been identified in Histoplasma. Mutations in these regulators render the pathogen unable to convert to the parasitic yeast form. Additionally, several virulence factors have been characterized for their importance in in vivo survival and pathogenesis. These virulence factors and regulators can serve as molecular handles for the development of effective drugs and therapeutics to counter Histoplasma infection.

Novel clinical and dual infection by Histoplasma capsulatum genotypes in HIV patients from Northeastern, Brazil.

Histoplasmosis is a worldwide-distributed deep mycosis that affects healthy and immunocompromised hosts. Severe and disseminated disease is especially common in HIV-infected patients. At least 11 phylogenetic species are recognized and the majority of diversity is found in Latin America. The northeastern region of Brazil has one of the highest HIV/AIDS prevalence in Latin America and Ceará State has one of the highest death rates due to histoplasmosis in the world, where the mortality rate varies between 33-42%. The phylogenetic distribution and population genetic structure of 51 clinical isolates from Northeast Brazil was studied. For that morphological characteristics, exoantigens profile, and fungal mating types were evaluated. The genotypes were deduced by a MSLT in order to define local population structure of this fungal pathogen. In addition, the relationships of H. capsulatum genotypes with clinically relevant phenotypes and clinical aspects were investigated. The results suggest two cryptic species, herein named population Northeast BR1 and population Northeast BR2. These populations are recombining, exhibit a high level of haplotype diversity, and contain different ratios of mating types MAT1-1 and MAT1-2. However, differences in phenotypes or clinical aspects were not observed within these new cryptic species. A HIV patient can be co-infected by two or more genotypes from Northeast BR1 and/or Northeast BR2, which may have significant impact on disease progression due to the impaired immune response. We hypothesize that co-infections could be the result of multiple exposure events and may indicate higher risk of disseminated histoplasmosis, especially in HIV infected patients.

Unravelling the interactions of the environmental host Acanthamoeba castellanii with fungi through the recognition by mannose-binding proteins.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.

Case Report: Hemophagocytic Lymphohistiocytosis Caused by Disseminated Histoplasmosis in a Venezuelan Patient with HIV and Epstein-Barr Virus Reactivation Who Traveled to Japan.

We describe a Venezuelan visitor to Japan who was diagnosed with hemophagocytic lymphohistiocytosis (HLH). The patient was also diagnosed with human immunodeficiency virus (HIV) and Epstein-Barr virus infection by the Western blot and polymerase chain reaction (PCR) tests, respectively. The cause of HLH was considered to be these two infections at first; however, the patient did not recover with antiretroviral/anti-herpes virus therapy. Thereafter, diagnosis of disseminated histoplasmosis was confirmed with an antigen detection test, culture, and PCR test of blood, urine, and bone marrow, and the patient improved gradually after the initiation of liposomal amphotericin B. This case highlights the importance of ruling out endemic mycosis as a cause of HLH even if other probable causes exist in patients from endemic areas.

Clinical outcomes and cortical reserve in adrenal histoplasmosis-A retrospective follow-up study of 40 patients.

OBJECTIVE: Detailed studies of Addison's disease resulting from disseminated adrenal histoplasmosis (AH) are not available. We describe the presentation and prognosis of AH and cortisol status before and after antifungal therapy. DESIGN: Single-centre retrospective hospital-based study of 40 consecutive adults with AH [39 males; age (mean ± SD) 53 ± 11 years] was conducted between 2006 and 2018. The median duration of follow-up was 2.5 years (range 0.2-12 years). PATIENTS AND METHODS: AH was diagnosed by bilateral adrenal enlargement on CT scan and presence of Histoplasma by histology and/or culture of biopsied adrenal tissue. All patients received oral itraconazole and, if required, amphotericin B as per guidelines. ACTH-stimulated serum cortisol (normal > 500 nmol/L) was measured in 38 patients at diagnosis and re-tested after one year of antifungal therapy in 21 patients. RESULTS: Seventy-three per cent of patients had primary adrenal insufficiency (PAI) and one-third had an adrenal crisis at presentation. HIV antibody was negative in all patients. Of the 29 patients who completed antifungal therapy, 25 (86%) were in remission at last follow-up. Overall, 8 (20%) patients died: three had a sudden death, four had severe histoplasmosis and one died due to adrenal crisis. No patient with PAI became eucortisolemic on re-testing after one year of antifungal therapy. Of the eight patients with normal cortisol at diagnosis, two developed adrenal insufficiency on follow-up. CONCLUSION: All patients with AH tested negative for HIV antibody. While patients achieved a high rate of clinical remission after antifungal therapy, overall mortality was significant. Cortisol insufficiency did not normalize despite treatment.