Survival in macrophages induces enhanced virulence in Cryptococcus.

Cryptococcus is a ubiquitous environmental fungus and frequent colonizer of human lungs. Colonization can lead to diverse outcomes, from clearance to long-term colonization to life-threatening meningoencephalitis. Regardless of the outcome, the process starts with an encounter with phagocytes. Using the zebrafish model of this infection, we have noted that cryptococcal cells first spend time inside macrophages before they become capable of pathogenic replication and dissemination. What "licensing" process takes place during this initial encounter, and how are licensed cryptococcal cells different? To address this, we isolated cryptococcal cells after phagocytosis by cultured macrophages and found these macrophage-experienced cells to be markedly more virulent in both zebrafish and mouse models. Despite producing a thick polysaccharide capsule, they were still subject to phagocytosis by macrophages in the zebrafish. Analysis of antigenic cell wall components in these licensed cells demonstrated that components of mannose and chitin are more available for staining than they are in culture-grown cells or cells with capsule production induced in vitro. Cryptococcus is capable of exiting or transferring between macrophages in vitro, raising the likelihood that this fungus alternates between intracellular and extracellular life during growth in the lungs. Our results raise the possibility that intracellular life has its advantages over time, and phagocytosis-induced alteration in mannose and chitin exposure is one way that makes subsequent rounds of phagocytosis more beneficial to the fungus.IMPORTANCECryptococcosis begins in the lungs and can ultimately travel through the bloodstream to cause devastating infection in the central nervous system. In the zebrafish model, small amounts of cryptococcus inoculated into the bloodstream are initially phagocytosed and become far more capable of dissemination after they exit macrophages. Similarly, survival in the mouse lung produces cryptococcal cell types with enhanced dissemination. In this study, we have evaluated how phagocytosis changes the properties of Cryptococcus during pathogenesis. Macrophage-experienced cells (MECs) become "licensed" for enhanced virulence. They out-disseminate culture-grown cells in the fish and out-compete non-MECs in the mouse lung. Analysis of their cell surface demonstrates that MECs have increased availability of cell wall components mannose and chitin substances involved in provoking phagocytosis. These findings suggest how Cryptococcus might tune its cell surface to induce but survive repeated phagocytosis during early pathogenesis in the lung.

Roles for Microglia in Cryptococcal Brain Dissemination in the Zebrafish Larva.

Cryptococcal infection begins in the lungs, but yeast cells subsequently access the bloodstream, from which they can reach the central nervous system (CNS). The resulting meningoencephalitis is the most common presentation and is very difficult to treat. How this fungus interacts with the blood-brain barrier (BBB) and establishes growth in the brain parenchyma remains a central question in fungal pathogenesis. We and others have developed the zebrafish larva as a model host for cryptococcosis and demonstrated that hematogenous CNS infection is replicated in this model. Here, we have used this model to examine the details of BBB crossing and the events immediately before and after. We have observed multiple mechanisms of BBB crossing and found that microglia, the resident phagocytes of the brain, likely have multiple roles. First, microglia either actively transfer yeast cells across the BBB or take up a significant proportion of them immediately after crossing. Second, microglia are capable of clearing individual cryptococcal cells at a developmental stage before adaptive immune cells have emerged. Third, microglia serve to maintain endothelial integrity, preventing other, phagocyte-independent forms of crossing. These proposed microglial functions during infection in the zebrafish larva generate new hypotheses concerning the establishment and control of cryptococcal meningoencephalitis. IMPORTANCE Cryptococcal meningitis is a fungal infection of the brain and a major cause of death in people with uncontrolled HIV. Infection begins in the lungs but can enter the bloodstream and disseminate to the brain. A structure called the blood-brain barrier must be crossed for the fungus to enter and cause meningitis. Learning how Cryptococcus crosses the blood-brain barrier will be crucial to understanding and possibly preventing brain infection. Using the zebrafish larva as a model host, we show that microglia, the resident phagocytes of the brain, potentially play multiple previously unappreciated roles in cryptococcal infection of the brain. These roles include reinforcing the integrity of the blood-brain barrier, clearing cryptococcal cells after they have crossed, and possibly participating directly in crossing via a previously unknown mechanism.

A Drosophila model of HPV16-induced cancer reveals conserved disease mechanism.

High-risk human papillomaviruses (HR-HPVs) cause almost all cervical cancers and a significant number of vaginal, vulvar, penile, anal, and oropharyngeal cancers. HPV16 and 18 are the most prevalent types among HR-HPVs and together cause more than 70% of all cervical cancers. Low vaccination rate and lack of molecularly-targeted therapeutics for primary therapy have led to a slow reduction in cervical cancer incidence and high mortality rate. Hence, creating new models of HPV-induced cancer that can facilitate understanding of the disease mechanism and identification of key cellular targets of HPV oncogenes are important for development of new interventions. Here in this study, we used the tissue-specific expression technique, Gal4-UAS, to establish the first Drosophila model of HPV16-induced cancer. Using this technique, we expressed HPV16 oncogenes E5, E6, E7 and the human E3 ligase (hUBE3A) specifically in the epithelia of Drosophila eye, which allows simple phenotype scoring without affecting the viability of the organism. We found that, as in human cells, hUBE3A is essential for cellular abnormalities caused by HPV16 oncogenes in flies. Several proteins targeted for degradation by HPV16 oncoproteins in human cells were also reduced in the Drosophila epithelial cells. Cell polarity and adhesion were compromised, resulting in impaired epithelial integrity. Cells did not differentiate to the specific cell types of ommatidia, but instead were transformed into neuron-like cells. These cells extended axon-like structures to connect to each other and exhibited malignant behavior, migrating away to distant sites. Our findings suggest that given the high conservation of genes and signaling pathways between humans and flies, the Drosophila model of HPV16- induced cancer could serve as an excellent model for understanding the disease mechanism and discovery of novel molecularly-targeted therapeutics.