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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., (© 2019 John Wiley & Sons Ltd.)

Most dimorphic fungal pathogens cause respiratory disease in mammals and must therefore possess virulence mechanisms to combat and overcome host pulmonary defenses. Over the past decade, advances in genetic tools have made it possible to investigate the basis of dimorphic fungal pathogenesis at the molecular level. Gene disruptions and RNA interference have now formally demonstrated the involvement of six virulence factors: CBP, alpha-(1,3)-glucan, BAD1, SOWgp, Mep1, and urease. Additional candidate virulence-associated genes have been identified on the premise that factors necessary for pathogenicity are associated specifically with the parasitic form. This principle continues to form the foundation for genomics-based analyses to further augment the list. Thus, the stage is set and the tools are in place for the next phase of medical mycology research: defining the virulence-associated factors underlying the success of dimorphic fungal pathogens.

The lipid content and composition of four strains of Paracoccidioides brasiliensis were analysed to determine any possible correlation with their virulence for hamsters and mice. Two strains, Pb168 and Pb141, were equal in virulence, Pb9 was slightly virulent and Pb140 was avirulent under the experimental conditions. No correlation was observed between virulence and the total lipid or phospholipid content of the strains. The lipid yield was highest in Pb9 and lowest in Pb168. Polar lipids were highest in Pb9 and least in Pb140. Phosphatidylcholine was the dominant phospholipid in all strains but its percentage was lower in the avirulent strain Pb140. Diphosphatidylglycerol, the least saturated lipid in all strains, was less abundant in Pb140 than in the virulent strains Pb168 and Pb141. In all four strains, neutral lipids constituted the major fraction of total lipids and triglycerides were the predominant individual lipid class, being more abundant in the avirulent and slightly virulent strains that in the virulent strains. The fatty acid profiles of total lipids and individual lipid classes of neutral and polar lipids obtained from the four strains were similar; however, the individual lipid classes showed patterns of preferential distribution of these fatty acids.

Groups of neotropical bats (Artibeus lituratus) were inoculated by the intraperitoneal or intranasal routes with varying doses of yeast phase Paracoccidioides brasiliensis. Bats infected with 10(6) viable yeast cells intraperitoneally developed fatal, disseminated disease, with delayed hypersensitivity appearing within 2 weeks. No precipitating antibodies were detected up to 7 weeks post-exposure. After intranasal instillation of 10(5) viable P. brasiliensis, the disease spread from the lung to the spleen by 3 weeks and to the liver by 9 weeks. As few as 10 viable cells were capable of causing pulmonary disease. Antibodies were detected at 5 weeks and persisted for several weeks thereafter. No viable P. brasiliensis was recovered from the intestines or fecal contents of any bats. Artibeus lituratus appears to be very susceptible to paracoccidioidomycosis by the respiratory route. The resulting immune response is characterized by delay appearance of precipitating antibodies and a moderate degree of delayed hypersensitivity. The pathogenesis of the resulting disease is similar to that observed in humans. The absence of intestinal involvement, even in chronic systemic disease, suggests that bats do not play a direct role in dissemination of this fungus in nature.

An adenine-requiring mutant of Paracoccidioides brasiliensis strain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Cell wall analysis of this mutant (strain IVIC Pb141) showed an increase in the amount of alpha-1,3-glucan and a virtual disappearance of the antigenic galactomannan. At the same time, a higher degree of virulence was observed for the mutant. These results agree with the hypothesis presented before (16) about a relationship between cell wall polysaccharides and pathogenicity in P. brasiliensis.

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis or South American blastomycosis. Many aspects of the disease and its agent are unknown. One of the most important factors regarding the infection and the host-parasite relationships seems to be the fungal cell wall whose biochemical aspects are reviewed here. Biochemical studies, done mainly by Kanetsuna et al., (21,22), have demonstrated that the yeastlike (Y) and the mycelial (M) forms have chitin as a common polysaccharide, with alpha-1, 3-glucan in the Y form and beta-1, 3-glucan in the M form. These polysaccharides are fibrillar and determine to some degree the fungal shape. Moreover, an amorphous galactomannan is found in the cell wall of the M form. This compound is responsible for the antigenic properties of the cell wall (1). Recent studies (30-33) suggest that the cell wall does not possess a stable chemical structure but a rather changing one, as a function of the environment in which the fungus is grown. At the same time, the cell wall composition seems to correlate with the degree of virulence of the particular strain. From these observations it may be deduced that the constituent polysaccharides of P. brasiliensis cell wall, play an important role in the active protection of the fungus against the defensive mechanisms of the host.

The yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9 reduced the amount of alpha-1,3-glucan in its cell wall from 45 to 3% when subcultured in vitro for several years. This strain regained its alpha-1,3-glucan up to 25% of the total cell wall when grown in vivo. A mutant strain of P. brasiliensis Pb9, named IVIC Pb140, reported to have 1,3-mannan instead of alpha-glucan in the cell wall, could not be recovered from experimentally infected animals. The existence of some relationship between the presence of alpha-1,3-glucan in the cell wall of the yeastlike form and the pathogenicity of this fungus is suggested in this report.

Virulence and infectivity of nine strains of Paracoccidioides brasiliensis were investigated in groups of mice which were inoculated intranasally or intravenously, and some of each were treated with corticosteroids. Fatal infections were not often seen among untreated mice, but mortality usually occurred when corticosteroids were given, regardless of the route of fungus inoculation. Prior treatment did not uniformly increase the incidence of infection, however; only in the case of intranasally inoculated mice was this effect seen. Most strains appeared to be more virulent when administered intravenously, with the exception of a single strain which, under the influence of corticosteroids, repeatedly displayed greatest virulence when given intranasally. All animals that died early in the course of the disease, irrespective of route of inoculation, always had acute pulmonary lesions and usually no other organ was involved. Animals which died later or were sacrificed always had chronic lung lesions. Whether or not chronically diseased animals had additional organ involvement correlated with how the organisms were administered; intravenously inoculated animals usually had extrapulmonary as well as pulmonary lesions, but lesions of those inoculated intranasally were almost exclusively pulmonary. Corticosteroids did not alter the histologic characteristics of either the acute or the chronic type of lesion, but the lesions of treated animals were usually more extensive. Most of the survivors appeared healthy even when infection was extensive.