Insertional mutagenesis enables cleistothecial formation in a non-mating strain of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Background  

Histoplasma capsulatum is a pathogenic ascomycete fungus that rapidly loses mating ability in culture. Loss of mating ability, as well as the organism's low rate of targeted gene replacement, limits techniques available for genetic studies in H. capsulatum. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in H. capsulatum strains, introducing a variety of classical genetics techniques to the field. We generated a strain, UC1, by insertional mutagenesis of the laboratory strain G217B, and found that UC1 acquired the ability to form mating structures called cleistothecia. The aim of this study was to determine the mechanism by which UC1 gained the ability to form cleistothecia. We also present initial studies demonstrating that UC1 can be used as a tool to determine molecular correlates of mating in H. capsulatum.     

Factors governing the epidemiology of Histoplasma capsulatum in soil

The growth ofH. capsulatum in soil is markedly affected by soil pH above 10 and below 5. No growth was observed on soil cultures outside these values. There was no definite pH range within these values in which the fungus grew more abundantly. A chemical analysis of natural soils from whichH. capsulatum had been isolated failed to show any common chemical factors that would explain the presence of the fungus in the soils.

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Zusammenfassung Das Wachstum desH. capsulatum im Erdboden ist durch das pH des Bodens über 10 und unter 5 wesentlich beeinflußt. Kein Wachstum ist in Bodenkulturen außerhalb dieser Werte beobachtet worden. Es fand sich keine bestimmte Grenze innerhalb dieser Werte, in welcher der Pilz ein üppigeres Wachstum gezeigt hätte. Eine chemische Analyse des naturlichen Bodens, von welchemH. capsulatum isoliert ist, hat keine gemeinsamen Faktoren gezeigt, der die Gegenwart des Pilzes im Boden hätte erklären konnen.

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Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

Cross-protection between Histoplasma capsulatum and Oidiodendron kalrai in mice

Summary Cross-protection studies were carried out by immunizing mice intraperitoneally with live and formalin killed yeast cells ofHistoplasma capsulatum andOidiodendron kalrai. Immunized and non-immunized mice were challenged intravenously 21 days later with the yeast cells ofHistoplasma capsulatum. The greatest protection was observed in mice immunized with live cells ofH. capsulatum and was definitely superior to that obtained with killed cells ofH. capsulatum. Significant protection against challenge byH. capsulatum was observed in mice immunized with killed but not with live cells ofO. kalrai.This work was supported from a research grant from the Bremer Foundation.The authors wish to thank Professor CharlotteC. Campbell for the supply ofHistoplasma capsulatum culture.     

Induction of novel protein synthesis by opsonizedHistoplasma capsulatum ingested by murine peritoneal macrophages

It is known thatHistoplasma capsulatum can resist the intraphagolysosomal environment and multiply inside macrophages. This resistance can be closly related to its pathogenicity. The mechanism of this resistance has been investigated, but it has not been clarified as yet. To learn about the metabolic condition of the yeast-form ofH. capsulatum (isolates G217B and CDC 105) when ingested by macrophages, we investigated protein synthesis by ingestedH. capsulatum with [³⁵S]-methionine labeling. Cycloheximide at 5 to 10 µg/ml was used to preferentially inhibit macrophage uptake of [³⁵S]-methionine without affectingH. capsulatum uptake. Protein synthesis byH. capsulatum in medium alone served as a positive control. The negative control consisted of macrophages with ingested heat-killedH. capsulatum. Analysis of cytosols with SDS-PAGE and fluorography disclosed that, respectively for G217B and CDC 105, ingestedH. capsulatum synthesized 4 and 5 novel proteins, increased the synthesis of 9 and 17 proteins and decreased the synthesis of 9 and 10 constitutive proteins. Ten of these novel or increased proteins were apparently common to both strains. These metabolic changes in ingestedH. capsulatum could reflect its adaptation to the intraphagolysosomal environment of macrophages and its ability to multiply there.     

Resistance of Histoplasma capsulatum to killing by human neutrophils

The basis for resistance of yeast form of Histoplasma capsulatum to antifungal activity of human neutrophils was studied. In limiting dilution assays and short term coculture assays human neutrophils were ineffective in killing H. capsulatum whereas Candida albicans was readily killed. By contrast, in a cell free hydrogen peroxide-peroxidase-halide system H. capsulatum was as sensitive to killing as C. albicans. Moreover, lysate of human neutrophils effectively substituted for horse-radish peroxidase in a cell free system for killing H. capsulatum. H. capsulatum elicited significant products of the oxidative burst in human neutrophils as detected by luminol-enhanced chemiluminescence. However, the response was two-fold less (p<0.05) than that induced by C. albicans. Transmission electron microscopy studies showed that phagosome-lysosome fusion took place when neutrophils phagocytosed C. albicans or H. capsulatum. Taken together, these findings indicate that, even though H. capsulatum elicits an oxidative burst and phagosome-lysosome fusion within the phagosome, it is capable of evading damage in short term assays.Abbreviations CFU colony forming units - PMN polymorphonuclear neutrophil - CTCM complete tissue culture medium - CL chemiluminescence - HPO horseradish peroxidase - P-L lysosomal peroxidase positive material     

Fluorescent antibody techniques for identification of Coccidioides immitis and Histoplasma capsulatum

This is a review of practical uses of immunofluorescence in detection of the two fungi in host and environment and in identification of their cultures, as well as in serologic case finding. Reagents directed at the yeast phase ofHistoplasma capsulatum have been fairly successful in differentiating this species from others, the main difficulty being the tendency to cross-react withBlastomyces dermatitidis andH. duboisii. Conjugates for the mycelial phase ofH. capsulatum tend to cross-react withSepedonium andChrysosporium, but careful absorption may yield specific reagents. Anti-yeast-phase conjugates are a valuable adjunct to cultural and clinical methods when used to detect and identifyH. capsulatum in sputum and other clinical specimens. Conjugates specific for the spherules or tissue phase ofCoccidioides immitis have yielded false negative results when applied to clinical specimens. The fluorescent-inhibition procedure is useful for serologic case finding in histoplasmosis and the same technique has shown fairly good agreement in coccidioidomycosis with complement-fixation and tube-precipitin methods. Immunofluorescence reagents for the two species have been useful in screening surgical and autopsy specimens, animal tissues, and soils.Paper read at the Eighth International Congresses for Tropical Medicine and Malaria, September 1968, Teheran (Iran).     

A survey of zoo aviaries for the presence of Histoplasma capsulatum and Cryptococcus neoformans

A zoo's aviaries were surveyed for the presence ofH. capsulatum andC. neoformans. Dried feces samples were collected in the 14 aviaries and tested forC. neoformans by using direct Cultural methods. This pathogen was isolated from an indoor aviary housing one White-breasted Toucan, and from another indoor aviary housing two Concave-casqued Hornbills. Soil samples were collected in 12 outdoor aviaries and two other areas of the zoo premises and tested forH. capsulatum andC. neoformans by direct and indirect cultural methods. Neither fungal pathogen was isolated from the soil samples. Fresh feces samples were collected from the two aviaries whereC. neoformans had been isolated in dried feces. Tests for the presence ofC. neoformans were made by direct culture methods, however the organism was not isolated.Contribution No. 154, Department of Infectious Diseases, College of Veterinary Medicine, Agricultural Experiment Station, Manhattan, 66502.     

II. Evidence of the presence in yeast extract of substances which stimulate the growth of Histoplasma capsulatum and blastomyces dermatitidis similarly to that found in starling manure extract

Summary A medium consisting of agar plus yeast extract contained the necessary metabolites for rapid growth and sporulation ofHistoplasma capsulatum andBlastomyces dermatitidis. H. capsulatum when harvested after 10 or 30 days incubation period from this medium was shown to have a similar number of spores as well as total particle viability for each period of growth.The growth characteristics ofH. capsulatum and four different isolates ofB. dermatitidis on yeast extract medium were similar to that obtained previously using starling (Sturnis vulgaris) manure extract medium. These characteristics are rapid growth consisting of many viable spores and a low ratio of vegetative mycelium.Several isolations ofH. capsulatum from naturally contaminated soil specimens were made using yeast extract medium.From the Communicable Disease Center, Public Health Service, U. S. Department of Health, Education, and Welfare.     

A clinical,pathological, and mycological study of a fatal case of histoplasmosis in an infant

Summary The clinical history of a fatal case of acute disseminated Histoplasmosis in a 3-month-old infant is described. A liver biopsy performed ante mortem revealed numerous encapsulated organisms which were identified at H.capsulatum. This suggests the applicability of this technique as a diagnostic tool. An autopsy showed extensive involvement, the organisms being found in the lungs, thymus, mediastinal and retroperitoneal nodes, liver, adrenals, spleen, and kidneys.A portion of emulsified liver was inoculated into Swiss mice andH. capsulatum was recovered from the mouse tissues 4 weeks after inoculation.A study was made of the cultural characteristic of this culture and it conformed to those previously described for other strains. A study of its fermentation properties disclosed no acid or gas production in media containing glucose, sucrose, lactose, maltose, mannose, rhamnose, trehalose, dulcitol, sorbitol, mannitol, and xylose.     

Comparative fluorescent antibody staining of histoplasma capsulatum and histoplasma duboisii with a specific anti-yeast phase H. Capsulatum conjugate

Summary The specific anti-yeast phaseHistoplasma capsulatum conjugate has been tested against 13 yeast phase strains ofH. capsulatum and 9 ofH. duboisii. The conjugate was specific forH. capsulatum, no yeast phase form ofH. duboisii obtained in vitro or in vivo reacted with it. The taxonomic implications of these results are discussed.     

The uptake of low molecular weight sulfur-containing compounds by Histoplasma capsulatum and related dimorphic fungi

A number of low molecular weight organic sulfur-containing compounds were tested for their effect on the respiratory activity of yeastlike and mycelialH. capsulatum. Of the compounds tested, L-cyst(e)ine was found to give maximum stimulatory effect on yeastlike phase respiration. The D- and meso isomers of cyst(e)ine as well as substituted derivatives were much less effective in the stimulation of respiratory activity of yeastlikeH. capsulatum. Respiration of homologous mycelial phase cell suspensions was depressed in the presence of L-cystine as substrate, while respiratory activity of yeastlikeB. dermatitidis andS. schenckii was unaffected.Whole cell suspensions of yeastlikeH. capsulatum actively transported S³⁵-labeled L-cystine and methionine but apparently not -mercaptoacetate-S³⁵. Mycelial phaseH. capsulatum and the yeastlike and mycelial phases ofB. dermatitidis andS. schenckii were observed to take up S³⁵-labeled L-cystine to a much lesser degree than yeastlikeH. capsulatum as determined on a dry weight basis. These results suggest significant differences in the transport and subsequent intracellular mechanisms of metabolism of low molecular weight sulfur-containing -amino acids and related compounds by yeastlikeH. capsulatum and its corresponding mycelial phase as well as the dimorphic fungiB. dermatitidis andS. schenckii.

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Zusammenfassung Eine Anzahl organischer, Sulfur-enthaltender Verbindungen mit niedrigem Molekulargewicht sind betreffs ihrer Wirkung an der Atmungsaktivität von hefeähnlichem und myzelialemH. capsulatum untersucht worden. Von den untersuchten Verbindungen gab L-cyst(e)ine die größte Reizwirkung an der Atmung der Hefephase. Die D- und Meso-Isomers von Cyst(e)ine so wie auch die substituierten Derivatives waren in der Reizung der Atmungsaktivität der Hefephase vonH. capsulatum weniger wirksam. Die Atmung der Suspension von Zellen der homologen Myzelphase war in der Gegenwart von L-cystine als Substrat unterdrückt, während die Atmungsaktivität der Hefephase desB. dermatitidis und die desS. schenckii unbeeinflußt blieb. Suspensionen von ganzen Zellen der Hefephase desH. capsulatum transportierten wirksam S³⁵ L-cystine und Methionine, aber anscheinend nicht beta-mercaptoacetate-S³⁵. Myzelphase-H. capsulatum und Hefeund Myzelphasen desB. dermatitidis undS. schenckii nehmen S³⁵-L-Cystine zu einem geringeren Grade auf denn Hefephase-H. capsulatum wie es am Trockengewicht festgestellt worden ist. Diese Ergebnisse legen es nahe, daß wesentliche Unterschiede im Transport und in dem nachfolgenden Intracellularmechanismus des Stoffwechsels von den Sulfurenthaltenden alfa-Aminosäuren mit niedrigem Molekulargewicht und verwandten Verbindingen durch die Hefephase desH. capsulatum und der bezüglichen Myzelphase, so wie auch durch die Doppelphasenpilze:B. dermatitidis undS. schenckii bestehen.

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This study has been supported by Part I VA-8200 Funds.     

Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires,Argentina

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

     

Estimation of the number of viable particles of Histoplasma capsulatum in soil

Summary Serial dilutions of suspensions of soil samples positive forH. capsulatum were made and injected intravenously into mice. The dilution producing infection in 50 % of the mice injected (ID₅₀) was determined for each sample and provided a measure for quantitative comparisons. A known number of viable particles ofH. capsulatum was added to soil, and serial dilutions were made of the suspension and injected into mice to determine that dilution containing an ID₅₀. One ID₅₀ was calculated to contain 1.6 viable particles ofH. capsulatum per ml of inoculum. With the assumption that one ID₅₀ of unknown samples contained 1.6 viable particles per ml inoculum, the total number of viable particles per gram of soil in several sites was calculated. The total number of viable particles ofH. capsulatum per gram of soil in different sites ranged from 101 to 201,900, almost a two thousandfold difference. Now that the number of viable particles ofH. capsulatum in positive sites can be determined, it may be possible to determine the concentration of particles necessary to make sites significant sources of infection.From the Ecological Investigations Program, National Communicable Disease Center, Bureau of Disease Prevention and Environmental Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Kansas City, Kansas.Presented in part at the annual meeting of the American Society for Microbiology, New York, N.Y., April 30-May 4, 1967.     

Is Histoplasma capsulatum a native inhabitant of Japan?

Histoplasmosis is an infectious disease caused by inhaling spores of the fungal pathogen H. capsulatum and in Japan is considered an imported mycosis. However, some patients in Japan with histoplasmosis have no history of traveling overseas nor of risk of occupational exposure to Histoplasma. To investigate the possibility of native distribution of Histoplasma in Japan, 187 bat guano samples from 67 bat‐inhabited caves in 17 prefectures were collected. These were examined for H. capsulatum by culture and Histoplasma‐specific PCR in three independent laboratories. No H. capsulatum was detected by either method, therefore H. capsulatum is unlikely to be present in bat guano in Japanese caves.     

The discovery ofHistoplasma capsulatum in Connecticut soil incidental to the investigation of a case of feline cryptococcosis

Summary The seventh case of cryptococcosis in a cat is described. The animal involved was a five-year-old Maltese short hair that had lived in Connecticut all of its life. An investigation was carried out to discover the point source of this feline infection. This search was unsuccessful but it did result in the isolation ofH. capsulatum for the first time from Connecticut soil.In addition, the preliminary findings of a study to evaluate the use of the fluorescent antibody technique for the rapid detection ofH. capsulatum in soil are reported. Round to oval forms measuring 1.5 to 3.5 microns in diameter were demonstrated in smears of the positive Connecticut soil that had been stained with fluorescein labeled anti-H. capsulatum globulins. The morphology of these elements was similar to that of the microconidia ofH. capsulatum. Through the use of this immune conjugate five additional positive soil samples from other areas were also shown to contain these forms. None of these six soils showed stained elements when treated with normal conjugates. The implications of these findings are discussed.     

The possible role of uric acid in the ecology of Histoplasma capsulatum

Summary The results of this study indicate thatHistoplasma capsulatum in its saprophytic form is able to utilize the major nitrogenous constituent of avian manure as a nitrogen source. In addition, the enzymes responsible for the pathway of uric acid degradation to inorganic nitrogen have been demonstrated in cell-free systems. These enzymes include uricase, allantoinase, allantoicase, and urease. The uricase ofHistoplasma appears to be a cell wall or cell membrane-associated enzyme, while the other enzymes were located in the soluble portion of cell-free extracts. Cell-free extracts ofCryptococcus neoformans are actively uricolytic.It is suggested that this ability ofH. capsulatum hyphae to utilize uric acid and related compounds as growth substrates may in part explain the indisputable ecologic association of this pathogenic fungus with avian and possibly chiropteran-associated soils and habitats in those areas endemic for histoplasmosis.From the Research Laboratories, Veterans Administration Hospital, Kansas City, Missouri, the Department of Biology, University of Missouri at Kansas City and the Department of Microbiology, University of Kansas School of Medicine, Kansas City, Kansas. Supported by VA-8200 funds.Portion of a Thesis presented by the senior author to the Graduate Faculty of the University of Missouri at Kansas City as partial fulfillment of the requirements for the Degree of Master of Arts.     

Environmental factors and growth of Histoplasma capsulatum in soil

Summary Environmental factors influencing the growth ofHistoplasma capsulatum in soil have been studied. The role of temperature and moisture in the growth of the fungus was found to be critical. The fungus can tolerate very low temperatures, if the soil moisture content is high, but cannot withstand temperatures of 40° C or above for an extended period.Dry, sterile chicken manure and an extract of unsterile chicken manure showed an inhibitory effect on the growth of the fungus. However, the relationship between bird manure andH. capsulatum has not been satisfactorily clarified.Morphological studies ofH. capsulatum in soil showed that the fungus grows within the upper two inches of the soil and a majority of sporulation occurs within the upper one-half inch of soil. Morphological studies of the fungus, in the presence of chicken manure and chicken manure extract, showed an increased number of macroconidia and microconidia and decreased mycelial production.The growth ofH. capsulatum in soil is markedly affected by soil pH above 10 and below 5. No growth was observed on soil cultures outside these values. There was no definite pH range within these values in which the fungus grew more abundantly.     

A study of fungi found in association with Histoplasma capsulatum: three bird roosts in S.E. Missouri,U.S.A.

Summary Soil samples from three bird roosts in SE Missouri were studied in an effort to detect differences in the distribution of microfungi in sites contaminated withHistoplasma capsulatum and presumably favorable, but uncontaminated sites. Seventy-four of 151 species identified were found in conjunction withH. capsulatum; many of the remaining 77 species were isolated only rarely and are probably not important colonizers of these soils. Only minor differences were noted regarding distribution of more commonly occurring species. Fungi known to be parasitic or antagonistic to other microfungi were about evenly distributed among the sites.H. capsulatum was most abundant in the one roost which was still occupied by flocks of birds.     

Soluble Components of <Emphasis Type="Italic">Histoplasma Capsulatum</Emphasis> var. <Emphasis Type="Italic">Capsulatum</Emphasis> have Hemagglutinin Activity and Induce Syngeneic Hemophagocytosis In Vitro

Histoplasma capsulatum var. capsulatum is a thermally dimorphic fungus that causes histoplasmosis. Fungal hemagglutination activity and cases of reactive hemophagocytic syndrome (RHS) have been reported in the disseminated form of disease. In the present study, soluble components of H. capsulatum var. capsulatum have been investigated for hemagglutinin activity and the capacity to induce hemophagocytosis in the mouse system. To analyze hemagglutinating activity, mouse red blood cells (RBC) (1% v/v in PBS) were incubated (37°C, 1 h) with cell-free antigen (CFAg) from H. capsulatum var. capsulatum (isolate IMT/HC128) (RBC-CFAg) or previously heated CFAg (56°C, 30 min) (RBC-hCFAg) or as control with PBS (RBC-PBS). Hemophagocytosis was analyzed by incubating BALB/c mouse peritoneal phagocytic cells (5 × 10⁶ cells) with syngeneic RBC, sensitized or not with CFAg. In addition, mouse polyclonal antibodies were raised against syngeneic RBC-CFAg (anti-RBC-CFAg) and used to analyze CFAg chromatographic fractions (Sephadex G75/120) by immunoenzymatic assay (ELISA). Hemagglutinin activity was observed with RBC-CFAg, but not with RBC-hCFAg or RBC. Also, hemophagocytosis was observed with RBC-CFAg, but not with RBC. The anti-RBC-CFAg antibodies reacted with CFAg fractions corresponding to a molecular mass (MM) higher than 150 kDa. In conclusion, the yeast form of H. capsulatum var. capsulatum releases thermolabile soluble components with hemagglutinin activity and it has been demonstrated for the first time that soluble components of the same fungus induce syngeneic hemophagocytosis in the in vitro mouse system. Also, indirect analysis with antibodies suggests that high-MM components (>150 kDa) are responsible for the interaction with RBC.     

Two Specific Strains of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> causing Mucocutaneous Manifestations of Histoplasmosis: Preliminary Analysis of a Frequent Manifestation of Histoplasmosis in Southern Brazil

Objectives  Skin lesions, uncommon in US cases (<10%), occur in 38–85% of cases reported from Latin America. Although these differences may reflect reporting bias, delayed diagnosis, or differences in host immune response among different ethnic groups, they also could result from genetic differences changing the pathobiology of the organism. It is possible that genetic differences among strains of H. capsulatum may influence the pathogenesis and clinical manifestations of histoplasmosis. Methods  We examined the clinical features of patients with mucocutaneous manifestations of histoplasmosis and performed genetic analysis based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes of H. capsulatum isolates of patients. Two pairs of PCR primers were designed to develop and amplify the ITS regions of H. capsulatum, 5′-TACCCGGCCACCCTTGTCTA-3′ and 5′-AGCGGGTGGCAAAGCCC-3′. These primers were based on the ITS sequence of Ajellomyces capsulatus, the ascomycetous teleomorph form of H. capsulatum, deposited in the GenBank (accession number U18363). Eight patients attending a tertiary-care hospital in southern Brazil were enrolled into the study. All case patients had skin cultures growing H. capsulatum at the mycology laboratory. Results  Six of eight (75%) patients were HIV-positive and presented involvement of multiples organs by H. capsulatum. Two HIV-negative patients did not present evidence of involvement of other organs besides mucosa and skin. ITS sequencing of a DNA H. capsulatum fragment of 485-bp from isolates of 8 patients revealed two distinct strains. The 2 distinct fragments (Hc1, Hc2) differed from each other at 7 positions in the ITS regions. They were identical to strains of H. capsulatum isolated in patients from Colombia and Argentina, but different from strains isolated in US. Hc1 and Hc2 were isolated in 5 patients and 3 patients, respectively, with mucocutaneous manifestations of histoplasmosis. Both Hc1 and Hc2 strains were isolated in HIV-infected and non-HIV-infected patients. Conclusions  Mucocutaneous manifestations of histoplasmosis, which are frequently seen in Brazilian patients were caused by 2 specific strains in our institution. Those strains have been isolated in patients with these particular clinical features of histoplasmosis in Latin America. Our study suggests that unique pathogenic characteristics among the Latin American species of H. capsulatum might explain its increased dermatotropism.