A Zebrafish Model of Cryptococcal Infection Reveals Roles for Macrophages, Endothelial Cells, and Neutrophils in the Establishment and Control of Sustained Fungemia

Abstract

Cryptococcal meningoencephalitis is a fungal infection that predominantly affects immunocompromised patients and is uniformly fatal if left untreated. Timely diagnosis is difficult, and screening or prophylactic measures have generally not been successful. Thus, we need a better understanding of early, asymptomatic pathogenesis. Inhaled cryptococci must survive the host immune response, escape the lung, and persist within the bloodstream in order to reach and invade the brain. Here we took advantage of the zebrafish larval infection model to assess the process of cryptococcal infection and disease development sequentially in a single host. Using yeast or spores as infecting particles, we discovered that both cell types survived and replicated intracellularly and that both ultimately established a sustained, low-level fungemia. We propose that the establishment and maintenance of this sustained fungemia is an important stage of disease progression that has been difficult to study in other model systems. Our data suggest that sustained fungemia resulted from a pattern of repeated escape from, and reuptake by, macrophages, but endothelial cells were also seen to play a role as a niche for cryptococcal survival. Circulating yeast collected preferentially in the brain vasculature and eventually invaded the central nervous system (CNS). As suggested previously in a mouse model, we show here that neutrophils can play a valuable role in limiting the sustained fungemia, which can lead to meningoencephalitis. This early stage of pathogenesis-a balanced interaction between cryptococcal cells, macrophages, endothelial cells, and neutrophils-could represent a window for timely detection and intervention strategies for cryptococcal meningoencephalitis. R ecent data from Uganda and Tanzania indicate that there are over 10,000 cases of HIV-related cryptococcosis per year in these countries alone (1, 2). Of those, roughly 5,000 to 8,000 (54 to 79%) are fatal. At a global scale, the survival rate is similarly poor, and over 600,000 deaths are attributed to Cryptococcus neoformans every year (3). The high mortality rate is in part due to difficulties in timely diagnosis; even in susceptible hosts, clinical signs of the initial pulmonary infection can be subtle or essentially absent (4). Studies in this population using serum cryptococcal antigen testing suggest that antigenemia (which may or may not represent actual fungemia) is a harbinger of meningoencephalitis but can precede it by weeks or more (5, 6). Once the infection has progressed to clinical meningoencephalitis, the prognosis is grim even with aggressive treatment. Beyond the HIV-infected population, a growing transplant and otherwise immunosuppressed population is also vulnerable, and even an immunocompetent host may be susceptible to infection and disease caused by the closely related species Cryptococcus gattii (7). An understanding of the events prior to fulminant disease is critical to better management of patients at risk for cryptococcal meningoencephalitis.While ...

Determining macrophage versus neutrophil contributions to innate immunity using larval zebrafish

“…In response to pathogens, macrophages can target and kill these microbes; for example, in response to the bacterial pathogen Salmonella, macrophages use LC3-associated phagocytosis to control this pathogen (Masud et al, 2019). But, in other infections, such as the bacterial pathogen B. cenocepacia (Mesureur et al, 2017) or the fungal pathogens A. fumigatus (Rosowski et al, 2018a), T. marneffei or the C. neoformans spore form (Davis et al, 2016), macrophages actually provide a protective niche for pathogen survival and growth. In sterile wounding conditions, macrophages can modulate the inflammatory microenvironment (Tsarouchas et al, 2018;Hasegawa et al, 2017;Nguyen-Chi et al, 2017), use Vegfa activation and mechanical forces to promote angiogenesis and vascular repair (Liu et al, 2016;Gurevich et al, 2018), and promote the repair and regrowth of damaged nerve tissue (Carrillo et al, 2016;Tsarouchas et al, 2018).…”

“…A different study of infection with the fungal pathogen C. neoformans found that when zebrafish larvae are infected with a less virulent strain and with spores instead of the yeast form of the pathogen, macrophages can play a pathogen-protective role (Davis et al, 2016). These fungal spores are phagocytosed by macrophages but later escape back into the vasculature.…”

“…These fungal spores are phagocytosed by macrophages but later escape back into the vasculature. Removal of this early intracellular niche in an irf8 mutant actually resulted in a lower fungal burden, although the increased neutrophil numbers in this genetic background may also contribute to fungal clearance (Davis et al, 2016).…”

Dragotcytosis: Elucidation of the Mechanism forCryptococcus neoformansMacrophage-to-Macrophage Transfer

“…Non-lytic exocytosis was implicated in persistence of fungemia(10) and may be involved in brain dissemination(23). Recently, non-lytic exocytosis was implicated as part of a process whereby phagocytic cells transfer C. neoformans to endothelial cells resulting in a new mechanism for cryptococcal crossing of the blood-brain barrier(16).…”

“…Cell-to-cell transfer is generally believed to be a process different from non-lytic exocytosis, with these two events being referred to as Type III and Type II exocytosis, respectively(9), denoting the fact that all these events share in common the exit of a fungal cell from an infected macrophage. Non-lytic exocytosis has been described in mammalian(7, 8), fish(10), insect(11), and amoeba(12) cells and appears to be a highly conserved strategy for C. neoformans cells to escape host and environmental predatory phagocytic cells. Non-lytic exocytosis has been described in other pathogenic microbes, including Burkholderia cenocepacia (13), Candida albicans (14), and Mycobacterium tuberculosis (15), suggesting that it may be a widespread strategy for microbial escape from phagocytic cells.…”