M. L. Taylor

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease ‘histoplasmosis’. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.

Histoplasma capsulatum yeast cells attach and agglutinate human erythrocytes

The ability of yeast cells of Histoplasma capsulatum to attach and agglutinate human erythrocytes has been described. This is the first report involving these yeasts in the hemagglutination phenomenon. Results revealed that the yeast cells were able to bind to erythrocytes irrespective of blood groups and to agglutinate them when a high density of yeast cells was used. Assays on the inhibition of yeast attachment to erythrocytes were also performed, using sugar-treated yeast cells. Results indicate that galactose (Gal), mainly the beta-anomer, specially inhibited yeast attachment. Disaccharides (Gal-derivatives) and glycosaminoglycans containing Gal residues, mainly chondroitin sulfate C, promote this type of inhibition. In addition, preliminary data of inhibition assays also involved a probable ionic strength driven mechanism mediated by sialic acid and heparan sulfate, suggesting that yeast binding to erythrocytes could be associated with negative charges of both molecules.

Characterizing Pneumocystis in the Lungs of Bats: Understanding Pneumocystis Evolution and the Spread of Pneumocystis Organisms in Mammal Populations

ABSTRACT Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.

Pneumocystis diversity as a phylogeographic tool

Parasites are increasingly used to complement the evolutionary and ecological adaptation history of their hosts. Pneumocystis pathogenic fungi, which are transmitted from host-to-host via an airborne route, have been shown to constitute genuine host markers of evolution. These parasites can also provide valuable information about their host ecology. Here, we suggest that parasites can be used as phylogeographic markers to understand the geographical distribution of intra-specific host genetic variants. To test our hypothesis, we characterised Pneumocystis isolates from wild bats living in different areas. Bats comprise a wide variety of species; some of them are able to migrate. Thus, bat chorology and migration behaviour can be approached using Pneumocystis as phylogeographic markers. In the present work, we find that the genetic polymorphisms of bat-derived Pneumocystis are structured by host chorology. Therefore, Pneumocystis intra-specific genetic diversity may constitute a useful and relevant phylogeographic tool.

An Hcp100 gene fragment reveals Histoplasma capsulatum presence in lungs of Tadarida brasiliensis migratory bats

Histoplasma capsulatum was sampled in lungs from 87 migratory Tadarida brasiliensis bats captured in Mexico (n=66) and Argentina (n=21). The fungus was screened by nested-PCR using a sensitive and specific Hcp100 gene fragment. This molecular marker was detected in 81·6% [95% confidence interval (CI) 73·4-89·7] of all bats, representing 71 amplified bat lung DNA samples. Data showed a T. brasiliensis infection rate of 78·8% (95% CI 68·9-88·7) in bats captured in Mexico and of 90·4% (95% CI 75·2-100) in those captured in Argentina. Similarity with the H. capsulatum sequence of a reference strain (G-217B) was observed in 71 Hcp100 sequences, which supports the fungal findings. Based on the neighbour-joining and maximum parsimony Hcp100 sequence analyses, a high level of similarity was found in most Mexican and all Argentinean bat lung samples. Despite the fact that 81·6% of the infections were molecularly evidenced, only three H. capsulatum isolates were cultured from all samples tested, suggesting a low fungal burden in lung tissues that did not favour fungal isolation. This study also highlighted the importance of using different tools for the understanding of histoplasmosis epidemiology, since it supports the presence of H. capsulatum in T. brasiliensis migratory bats from Mexico and Argentina, thus contributing new evidence to the knowledge of the environmental distribution of this fungus in the Americas.

Relationship between age and cellular suppressive activity in resistance to Histoplasma capsulatum infection

One-month-old and 1-year-old male BALB/c mice showed a lower resistance than 4.5-month-old mice to Histoplasma capsulatum infection. 4.5-month-old mice successfully resolved the infection when challenged with either a LD50 or LD100 for 1-month-old mice. A critical clinical course of experimental histoplasmosis was observed in 4.5-month-old syngeneic mice when spleen cells from 1-month-old BALB/c mice were transferred to them. Irradiated recipient mice, into which bone marrow and spleen cells were transferred, died when infected with the LD100 for 1-month-old mice. The same occurred with 4.5-month-old non-irradiated infected mice which received only spleen cells and with 1-month-old mice which were used as a control of infection. However, infected and non-transferred 4.5-month-old mice survived this dose. Thus, the adoptive transference of spleen cells from 1-month-old mice to 4.5-month-old mice suppressed the resistance of these adult mice to infection. Apparently, the transference of the suppressive state requires the presence of two cell populations, a non-adherent and an adherent and radioresistant cell present in the spleen of male 1-month-old mice.

Frequency and Genetic Diversity of the MAT1 Locus of Histoplasma capsulatum Isolates in Mexico and Brazil

ABSTRACT The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+ mating type) and G-186AR (− mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective + and − mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources.