Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.     

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.     

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.     

Intraspecific chromosomal variability in human pathogenic fungi, especially in Histoplasma capsulatum

The ploidy, karyotype, and chromosome length polymorphism (CLP) of human pathogenic fungi were revised with emphasis on Histoplasma capsulatum, the causative agent of the systemic mycosis, histoplasmosis. Currently, different systems of gel electrophoresis are being used to determine fungal electrokaryotypes (EK). By renaturation kinetic and genomic reconstruction in H. capsulatum strains (G-186AS and Downs), estimated genome sizes of 23 and 32 Mb were determined for both strains, respectively. The haploid state was proposed for both strains, although aneuploidy was suggested for the Downs strain. Contour-clamped homogeneous electric field (CHEF), field inversion gel electrophoresis (FIGE), and Southern blot using different probes showed the presence of six to seven chromosomes in the Downs strain (low virulence), whereas four chromosomes were identified in the G-186B strain (high virulence). The use of these methods in the three major H. capsulatum reference strains (G-217B and Downs from the United States of America, G-186B from Panama) revealed distinct chromosome sizes, from 0.5 to 5.7 Mb, with CLP associated with chromosomes size and mobility. Recently, by CHEF, using 19 H. capsulatum isolates from Latin-America and the G-186B strain, five to seven chromosomes with 1.1 to 11.2 Mb molecular sizes were revealed, which again suggested CLP in H. capsulatum. However, to elucidate the EKs polymorphism in H. capsulatum and its relationship with the isolates phenotype more studies are needed to understand the mechanisms controlling ploidy variability.     

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.

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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.

     

Surface localization of the Yps3p protein of Histoplasma capsulatum

The YPS3 gene of Histoplasma capsulatum encodes a protein that is both resident in the cell wall and also released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differently in H. capsulatum strains that differ in virulence. We investigated the cellular localization of Yps3p. We demonstrated that the cell wall fraction of Yps3p was surface localized in restriction fragment length polymorphism class 2 strains. We also established that Yps3p released into the G217B culture supernatant binds to the surface of strains that do not naturally express the protein. This binding was saturable and occurred within 5 min of exposure and occurred similarly with live and heat-killed H. capsulatum. Flow cytometric analysis of H. capsulatum after enzymatic treatments was consistent with Yps3p binding to chitin, a carbohydrate polymer that is a component of fungal cell walls. Polysaccharide binding assays demonstrated that chitin but not cellulose binds to and extracts Yps3p from culture supernatants.     

Phylogeography of the fungal pathogen Histoplasma capsulatum

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein‐coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein‐coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.     

Cysteine-independent and cysteine-requiring yeast-strains of Histoplasma capsulatum

Recently we described a strain of Histoplasma capsulatum, designated H-35, which is able to grow as yeast on a minimal medium consisting of inorganic salts, glucose and a trace of biotin. Using this strain as a prototrophic wild type we sought auxotrophic mutants. Mutagenized yeast-cells were starved for inorganic sulfate in sulfur-free minimal medium. Sulfate was then added, and growing prototrophic cells were killed by addition of amphotericin B. After 24 hours non-growing auxotrophs were rescued by removal of amphotericin and addition of yeast extract. This mutant enrichment cycle was repeated two additional times, after which the cells were plated on blood agar and 800 yeast-colonies were picked. Seventeen of these yeast-strains required cysteine for growth, as compared with strain H-35, which grew as yeast on minimal medium.     

The dimorphism and infectivity of Histoplasma capsulatum

Summary By examining mycelial cultures at regular intervals during incubation at 37° C in cysteine-enriched media, it was possible to detect differences in the conversion properties of six isolates ofHistoplasma capsulatum. The organisms varied according to conversion ability, rate and degree of transformation following yeast initiation, and cysteine sensitivity. These findings were unrelated to infectivity of the mycelial phase for mice as determined by percentage recovery of isolates from the cultured organs of inoculated animals.     

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.