The long-term goal of this proposal is to study mechanism(s) of pathogenicity of Cryptococcus neoformans (Cn)focusing on how Cn sphingolipids regulate fungal virulence and counteract the host immune response.
Cn is a fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life-threatening meningo-encephalitis in susceptible patients. One key feature of Cn is its ability to grow both extracellularly and intracellularly. Being the lung the port of entry of Cn, alveolar macrophages (AMs) are the first line of defense of the host. Thus, the outcome of the interaction between the fungus and AMs greatly determines the progression of the disease. If the immune system of the host is compromised and AMs fail to kill the engulfed Cn cells, these fungal cells survive and proliferate within the phagolysosome of AMs. This implies that the physiopathology of cryptococcosis is determined by fungal growth that can occur in an intracellular compartment (e.g. acidic environment of AM’s phagolysosome) and in an extracellular compartment (e.g. neutral/alkaline environment of alveolar spaces and bloodstream).
In the past funding cycle, we identified novel fungal factors in the sphingolipid pathway that specifically regulate growth of Cn in each compartment. Particularly, the inositol phosphosphingolipid phospholipase C (Isc1) enzyme was found to be required for intracellular growth of Cn within AMs, whereas the glucosylceramide synthase (Gcs1) was found to be required for extracellular growth of Cn within the alveolar spaces. Importantly, we identified very long chain C26 phytoceramide and glucosylceramide (GlcCer) as the biochemical products of the reactions catalized by Isc1 and Gcs1, respectively. Thus, we hypothesize that Isc1 and Gcs1 regulate pathogenicity by favoring growth in different compartment(s) through the action of the specific sphingolipids that they produce. This hypothesis will be addressed by the following Aims: 1) To study the mechanism by which fungal sphingolipids regulate growth of Cn in the intracellular compartment; 2) To study the mechanism by which fungal sphingolipids regulate growth of Cn in the lung-like extracellular compartment; 3) To determine the therapeutic effect of targeting intracellular and/or extracellular populations of Cn on the outcome of the infection.
Role of phagocytosis in the pathogenesis of Cryptococcus neoformans
The long-termgoal of this project is to elucidate the molecular mechanism(s) controlling the phagocytic process of the pathogenic fungus Cryptococcus neoformans (Cn) by host alveolar macrophages (AMs). Understanding these regulatory mechanisms will reveal new therapeutic strategies for the attenuation of the disease process. Cn is an opportunistic and facultative intracellular fungal pathogen that infects humans via the respiratory tract. Dissemination of the infection leads to development of a life-threatening meningo-encephalitis, particularly in immunocompromised patients. Phagocytosis of Cn by AMs represents the first line of defense by the host, and the killing of the organism is controlled by an efficient host-cell response. However, in conditions of cellular immune deficiency, phagocytosis may become detrimental for the host because Cn can grow and disseminate within macrophages. Thus, internalization of Cn by phagocytic cells may be considered either an obstacle or an opportunity for disease development, and fungal factors that control the phagocytic process may assume a crucial role in the outcome of the infection.
We identified a novel cryptococcal gene encoding for an antiphagocytic protein 1 (App1), which inhibits phagocytosis of Cn by AMs. A Cn mutant lacking App1 is less pathogenic in a mouse model with a functional host-cell response but, intriguingly, more pathogenic in mice with impaired host-cell response compared to Cn wild-type strain. These observations lead us to hypothesize that App1 modulates pathogenicity of Cn through the regulation of phagocytosis by AMs. This hypothesis will be tested by the following Specific Aims: 1) Determine the mechanisms by which App1 regulates phagocytosis, and 2) determine the role and function of App1 in the outcome of cryptococcosis. These studies will produce new insights into the mechanisms of pathogenicity of C. neoformans at the host-microbe interface. Importantly, these studies will potentially provide new therapeutic approaches to better control the development of cryptococcal infection.
Host Sphingolipids and Fungal Infections
The goal of this project is to study the role and mechanisms by which host sphingolipids are involved in controlling the infection caused by the pathogenic fungus Cryptococcus neoformans (Cn).
A rapidly emerging area of research is the study of the role of sphingolipids in the regulation of infectious diseases. Most of these studies have focused on the role of microbial sphingolipids in the ability of the microbe to cause infection. Very few studies addressed if and how host sphingolipids are also involved in the regulation of microbial pathogenesis, and most of these studies have focused on bacterial or parasitic infections. Although some sphingolipids have been linked to antibacterial activity of phagocytic cells nothing is known about the role of host sphingolipids against fungal infections. Since phagocytic cells, such as macrophages and neutrophils, are the first line of defense against Cn infection, the regulation of their cellular processes may affect their response to the fungus, and thus, determine whether they can or cannot control the development of cryptococcosis.
One of the host sphingolipid-metabolizing enzymes shown to regulate immune responses is sphingomyelin synthase (SMS) encoded by two genes, SMS1 and SMS2. SMS transfers a choline phosphate moiety from phosphatidylcholine (PC) to ceramide, therefore producing sphingomyelin (SM) and diacylglycerol (DAG). Very interestingly, the lipids regulated by SMS have been implicated in the activation of pro-inflammatory responses, suggesting that the regulation of SMS activity in immune cells may assume a critical role in controlling infections.
In our preliminary studies we found that: 1) inhibition of SMS activity profoundly impairs the ability of neutrophils to kill Cn cells by affecting extracellular killing in absence of phagocytosis; 2) SMS regulates production of DAG at the Golgi; 3) DAG at the Golgi regulates protein secretion through a protein kinase D (PKD)-mediated mechanism; 4) inhibition of PKD blocks extracellular killing of Cn; and 5) inhibition of either SMS or PKD activity significantly decreases secretion of antimicrobial peptite(s) such as a-defensin. Based on these observations, we hypothesize that SMS activity plays a key role in controlling Cn infection through a DAG-PKD-antimicrobial peptide(s) secretion pathway. Thus, we propose the following aims:
1) To determine the role of SMS activity during Cn infection; and 2) To define the mechanism by which SMS activity regulates the extracellular killing of Cn.
By studying how host sphingolipids regulate the extracellular killing activity of neutrophils against Cn, we will provide new insights not only for a better understanding of fungal pathogenesis but also for the development of new therapeutic strategies against this and potentially other fungal microbes. The studies that we propose in this application will reveal new regulatory mechanisms involved in the killing of Cn by neutrophils. We will identify the nexus between SMS, PKD and infection.