Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

Conservation of Intracellular Pathogenic Strategy among Distantly Related Cryptococcal Species.

The genus Cryptococcus includes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to be Cryptococcus neoformans and Cryptococcus gattii We compared the interaction of murine macrophages with three C. gattii species complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and one C. neoformans species complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis. The most striking result was that the intracellular pathogenic strategies of C. neoformans and C. gattii species complex strains were qualitatively similar, despite the species having separated an estimated 100 million years ago. Macrophages exhibited a leaky phagolysosomal membrane phenotype and nonlytic exocytosis when infected with either C. gattii or C. neoformans Conservation of the intracellular strategy among species that separated long ago suggests that it is ancient and possibly maintained by similar selection pressures through eons.

Cryptococcus neoformans releases proteins during intracellular residence that affect the outcome of the fungal-macrophage interaction.

Cryptococcus neoformans is a facultative intracellular pathogen that can replicate and disseminate in mammalian macrophages. In this study, we analyzed fungal proteins identified in murine macrophage-like cells after infection with C. neoformans. To accomplish this, we developed a protocol to identify proteins released from cryptococcal cells inside macrophage-like cells; we identified 127 proteins of fungal origin in infected macrophage-like cells. Among the proteins identified was urease, a known virulence factor, and others such as transaldolase and phospholipase D, which have catalytic activities that could contribute to virulence. This method provides a straightforward methodology to study host-pathogen interactions. We chose to study further Yeast Oligomycin Resistance (Yor1), a relatively uncharacterized protein belonging to the large family of ATP binding cassette transporter (ABC transporters). These transporters belong to a large and ancient protein family found in all extant phyla. While ABC transporters have an enormous diversity of functions across varied species, in pathogenic fungi they are better studied as drug efflux pumps. Analysis of C. neoformans yor1Δ strains revealed defects in nonlytic exocytosis, capsule size, and dimensions of extracellular vesicles, when compared to wild-type strains. We detected no difference in growth rates and cell body size. Our results indicate that C. neoformans releases a large suite of proteins during macrophage infection, some of which can modulate fungal virulence and are likely to affect the fungal-macrophage interaction.

Breast cancer subtypes express distinct receptor repertoires for tumor-associated macrophage derived cytokines.

Infiltration of the tumor microenvironment by macrophages is associated with poor outcomes in breast cancer and other solid tumors, however the identity and roles of many of the soluble factors these macrophages produce remains to be elucidated in detail. In addition to producing angiogenic factors (e.g. VEGF), proteases (e.g. MMP9) and immunomodulatory factors (e.g. IL10) which, by modifying the local microenvironment, likely contribute to progression in the majority of solid tumors, we have evaluated the extent to which macrophage cytokines may differentially affect distinct breast cancer subtypes. We identified 23 cytokines produced in a culture model of human tumor-associated macrophages and report that basal and luminal breast cancer cell lines express different repertoires of receptors for these cytokines. These data suggest that tumor-associated macrophages make specific contributions to different breast cancer subtypes and that understanding the importance of these interactions will be crucial to developing subtype-specific therapies targeting the macrophage component of the breast tumor microenvironment.

Novel Antifungal Compounds Discovered in Medicines for Malaria Venture's Malaria Box.

Similarities in fungal and animal cells make antifungal discovery efforts more difficult than those for other classes of antimicrobial drugs. Currently, there are only three major classes of antifungal drugs used for the treatment of systemic fungal diseases: polyenes, azoles, and echinocandins. Even in situations where the offending fungal organism is susceptible to the available drugs, treatment courses can be lengthy and unsatisfactory, since eradication of infection is often very difficult, especially in individuals with impaired immunity. Consequently, there is a need for new and more effective antifungal drugs. We have identified compounds with significant antifungal activity in the Malaria Box (Medicines for Malaria Ventures, Geneva, Switzerland) that have higher efficacy than some of the currently used antifungal drugs. Our best candidate, MMV665943 (IUPAC name 4-[6-[[2-(4-aminophenyl)-3H-benzimidazol-5-yl]methyl]-1H-benzimidazol-2-yl]aniline), here referred to as DM262, showed 16- to 32-fold-higher activity than fluconazole against Cryptococcus neoformans. There was also significant antifungal activity in other fungal species with known antifungal resistance, such as Lomentospora prolificans and Cryptococcus gattii. Antifungal activity was also observed against a common fungus, Candida albicans. These results are important because they offer a potentially new class of antifungal drugs and the repurposing of currently available therapeutics. IMPORTANCE Much like the recent increase in drug-resistant bacteria, there is a rise in antifungal-resistant strains of pathogenic fungi. There is a need for novel and more potent antifungal therapeutics. Consequently, we investigated a mixed library of drug-like and probe-like compounds with activity in Plasmodium spp. for activity against two common fungal pathogens, Cryptococcus neoformans and Candida albicans, along with two less common pathogenic species, Lomentospora prolificans and Cryptococcus gattii. We uncover a previously uncharacterized drug with higher broad-spectrum antifungal activity than some current treatments. Our findings may eventually lead to a compound added to the arsenal of antifungal therapeutics.

Mutant GATA3 Actively Promotes the Growth of Normal and Malignant Mammary Cells.

BACKGROUND/AIM: GATA3, a transcription factor expressed in luminal breast epithelial cells, is required for mammary gland development. Heterozygous GATA3 mutations occur in up to 15% of estrogen receptor (ER)-positive breast tumors and have been proposed to be null alleles resulting in haploinsufficiency; however, the mutation spectrum of GATA3 in breast cancer is in sharp contrast to that found in HDR syndrome, a true GATA3 haploinsufficiency disease. MATERIALS AND METHODS: Transgenic mice, 3D cultures and xenografts were used to examine the effect of mutant GATA3 expression on mammary cell proliferation. RESULTS: Mutant GATA3 accelerated tumor growth of ZR751 cell xenografts and promoted precocious lobuloalveolar development in transgenic mouse mammary glands. CONCLUSION: GATA3 mutations, recently observed in breast cancer, encode active transcription factors, which elicit proliferative phenotypes in normal mammary epithelium and promote the growth of ER-positive breast cancer cell lines.