Histoplasma hairpins herald hopefulness

Histoplasma capsulatum is a significant respiratory and systemic fungal pathogen. Although many molecular tools have been developed, the fulfillment of Koch's postulates to determine gene function has been hampered by obstacles to homologous gene targeting. Because H. capsulatum displays a considerable array of virulence mechanisms and has a 40-Mb genome that is currently being sequenced, the capability to perform high-throughput molecular manipulations would clearly be beneficial. Recent demonstration and application of experimental RNA interference (RNAi) technology promises a major contribution to advances in this area.     

Selenocystine-resistant mutants of Histoplasma capsulatum

Cysteine metabolism has been thought to be important to the phenomenon of dimorphism inHistoplasma capsulatum. We sought mutants with genetic blocks in the metabolism of cysteine by selection of colonies resistant to the toxic analogue, selenocystine. The 22 resistant strains thus obtained were all deficient in uptake of cystine from the surrounding medium but were normally able to convert from mycelium to yeast and back again. Furthermore, they had normal quantities of NADH-dependent cystine reductase when this enzyme was measured. We conclude that mutants defective in cystine uptake can be readily obtained by selection of colonies resistant to selenocystine, and that a lesion in cystine-uptake does not appear to affect the phenomenon of dimorphism in this organism.Preliminary reports of this work were presented at the Second International Congress of Mycology, Tampa, 1977 and at the first International Conference on Histoplasmosis, Atlanta, 1978.     

Chemical composition of Histoplasma capsulatum

The chemical composition of yeast and mycelial cells of three strains ofHistoplasma capsulatum was analyzed and is expressed as per cent dry weight. Cultures were grown in a liquid synthetic medium, mycelial cells at 25°C and yeast at 37°C on gyrotory shakers. After 7 days, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were higher in the yeast cells while mycelial cells contained more lipid and carbohydrate. The components of one strain were also studied at different stages of growth. The DNA in both yeast and mycelial cells remained relatively constant, but other components varied with the age of culture. In yeast cells the RNA level was 6.8 % at 2 days and then declined sharply remaining constant around 3.5 %. A protein content of 29 % on day 2 decreased gradually to 19 % on day 14. An initial lipid content of 21 % rose to 33 % on day 5 and then decreased. Similarly, an initial carbohydrate level of 17 % rose to 25 % on day 7 and then declined. The mycelial cells contained 4 % RNA up to 10 days followed by a slight decline to 3 % on day 14. A protein content of 20 % on day 5 increased to 24 % on day 10 and then decreased to 15 % on day 28. The lipid content of 33 % on day 5 rose to 38 % on day 7 and then decreased gradually. The carbohydrate level of 20 % at 5 days increased to 38 % on day 10 and declined gradually to 27 % after 28 days.

---

Résumé La composition chimique des cellules levuriformes et mycéliennes de trois souches deHistoplasma capsulatum a été déterminée. Le champignon a été cultivé dans un milieu synthétique liquide secoué à 25° C pour la phase mycélienne et à 37° C pour la phase levuriforme. Après 7 jours d'incubation, les cellules levuriformes étaient plus riches en acides nucléiques et en protéines que les cellules mycéliennes qui étaient par contre plus riches en lipides et en hydrates de carbone. La composition d'une des souches fut étudiée à différentes étapes de la croissance. La teneur en ADN des deux phases resta relativement constante mais des variations furent observées dans le cas des autres constituants chimiques. Pour ce qui est des levures, l'ARN qui constituait 6,8 % du poids des cellules sèches à deux jours, tomba rapidement à 3,5 % et resta constant. Les proteines passèrent de 29 % au deuxième jour à 19 % au quatorzième jour. Au contraire, la teneur en lipides passa d'un valeur initiale de 21 % à 33 % au cinquième jour, pour diminuer de nouveau par la suite. De même, une teneur initiale en hydrates de carbone de 17 % passa à 25 % au septième jour puis diminua par la suite. Dans les cas des cellules mycéliennes contenaient 4 % de ARN jusqu'au dizième jours, puis cette valeur tomba légèrement jusqu'à 3 % au quatorzième jour. Les protéines qui représentaient 20 % au cinquième jour augmentèrent jusqu'à 24 % au dizième jour pour tomber à 15 % au vingthuitième jour. La teneur en lipides de 33 % au cinquième jour augmenta jusqu'à 38 % au septième jour pour diminuer graduellement. De même les taux en hydrates de carbones qui représentaient 20 % au cinquième jour augmentèrent jusqu'à 38 % au dixième jour et diminuèrent graduellement jusqu'à 27 % au vingt-huitième jour.

     

Characterization of the Histoplasma capsulatum-induced granuloma

Rising rates of Histoplasma capsulatum infection are an emerging problem among the rapidly growing population of immune-compromised individuals. Although there is a growing understanding of systemic immunity against Histoplasma, little is known about the local granulomatous response, which is an important component in the control of infection. The focus of this article is the characterization of Histoplasma-induced granulomas. Five days after i.p. infection, infected macrophage appear in the liver and lung; however, no granulomas are apparent. Two days later, well-formed sarcoid granulomas are abundant in the lung and liver of infected mice, which contain all visible Histoplasma. Granulomas are dominated by macrophage and lymphocytes. Most of the Histoplasma and most of the apoptotic cells are found in the center of the lesions. We isolated liver granulomas at multiple time points after infection and analyzed the cellular composition, TCR gene usage, and cytokine production of granuloma-infiltrating cells. The lesions contain both CD4+ and CD8+ T cell subsets, and T cells are the primary source of IFN-gamma and IL-17. The main source of local TNF-alpha is macrophage. Chemokines are produced by both infiltrating macrophage and lymphocytes. Dendritic cells are present in granulomas; however, T cell expansion seems to occur systemically because TCR usage is very heterogeneous even at the level of individual lesions. This study is the first direct examination of host cellular responses in the Histoplasma-induced granuloma representing the specific interface between host and pathogen. Our studies will allow further analysis of key elements of host Histoplasma interactions at the site of chronic infection.     

A prototrophic yeast-strain of Histoplasma capsulatum

A newly derived strain of Histoplasma capsulatum can be grown stably as yeast in a minimal medium containing glucose, biotin, tartrate and inorganic salts.     

Inhibition of Histoplasma capsulatum by Garlic

The mycelial phase ofHistoplasma capsulatum was inhibited by both the volatile and water soluble components of garlic,Allium sativum L. Garlic extract at a concentration of 254 parts per billion (ppb) was inhibitory, while 8.1 parts per million (ppm) were lethal to pure cultures ofH. capsulatum. The role of garlic as an eradicent is discussed.The work was conducted while the author was a graduate student at the University of Kentucky, Lexington, Kentucky.     

Isolation of aminopeptidases from Histoplasma capsulatum

Two aminopeptidases (arylamidases) were isolated and partially purified from Histoplasma capsulatum. The larger molecular weight enzyme was a proline iminopeptidase and hydrolyzed primarily a synthetic substrate, L-prolyl-beta-napthylamide. The other aminopeptidase was less substrate specific and hydrolyzed rapidly the following amino acid beta-napthylamides (beta NA): L-arginyl-beta NA greater than L-lysyl-beta NA greater than -L-4-methoxy-leucyl-beta NA greater than L-leucyl-beta NA greater than L-phenylalanyl-beta NA greater than L-alanyl-beta NA. The proline iminopeptidase was purified 1420 fold while the leucine aminopeptidase was purified 650 fold with good recovery.     

Histoplasma capsulatum at the host-pathogen interface

Histoplasma capsulatum is the most common cause of invasive fungal pulmonary disease worldwide. The interaction of H. capsulatum with a host is a complex, dynamic process. Severe disease most commonly occurs in individuals with compromised immunity, and the increasing utilization of immunomodulators in medicine has revealed significant risks for reactivation disease in patients with latent histoplasmosis. Fortunately, there are well developed molecular tools and excellent animal models for studying H. capsulatum virulence and numerous recent advances have been made regarding the pathogenesis of this fungus that will improve our capacity to combat disease.     

Development and Use of a Polyvalent Conjugate to Differentiate Histoplasma capsulatum and Histoplasma duboisii from Other Pathogens

Previous investigations have demonstrated the existence of five Histoplasma capsulatum serotypes. Available specific fluorescent-antibody reagents stain only four of the five serotypes. Antibodies produced against the most complete H. capsulatum serotype were labeled with fluorescein isothiocyanate to develop a reagent specific for H. capsulatum that was reactive with all the known serotypes. The unadsorbed reagent not only stained all the H. capsulatum serotypes, but it also stained cultures of Blastomyces dermatitidis, H. duboisii, several Candida species, and a variety of other fungi. Adsorption of the conjugate with antigens of C. albicans produced a reagent that intensely stained only H. capsulatum, H. duboisii, and B. dermatitidis. Differentiation of B. dermatitidis from the Histoplasma species was accomplished by application of a B. dermatitidis specific fluorescent antibody to antigens positive with the H. capsulatum reagent. At present, differentiation of H. capsulatum from H. duboisii may be accomplished only by animal inoculation. Our data substantiate the antigenic relationships hypothesized earlier, and they indicate that H. capsulatum shares at least two antigens with the other fungi that were studied.